VERMONT STEM CELL CONFERENCE Stem Cells and Cell Therapies in Lung Biology and Diseases: Conference Report Daniel J. Weiss, Jason H. T. Bates, Thomas Gilbert, W. Conrad Liles, Carolyn Lutzko, Jay Rajagopal, and Darwin Prockop

Executive Summary The University of Vermont College of Medicine and the Vermont Lung Center, with support of the National Heart, Lung, and Blood Institute (NHLBI), the Alpha-1 Foundation, the American Thoracic Society (ATS), the LAM Treatment Alliance, and the Pulmonary Fibrosis Foundation, convened a workshop, “Stem Cells and Cell Therapies in Lung Biology and Lung Diseases,” held July 25 to 28, 2011 at the University of Vermont, to review the current understanding of the role of stem and progenitor cells in lung repair after injury and to review the current status of cell therapy and ex vivo bioengineering approaches for lung diseases. These are rapidly expanding areas of study that both provide further insight into and challenge traditional views of mechanisms of lung repair after injury and pathogenesis of several lung diseases. The goals of the conference were to summarize the current state of the field, discuss and debate current controversies, and identify future research directions and opportunities for both basic and translational research in cell-based therapies for lung diseases. This conference was a follow-up to three previous conferences held at the University of Vermont: “Adult Stem Cells, Lung Biology, and Lung Disease” sponsored by the NHLBI, the Cystic Fibrosis Foundation, the University of Vermont College of Medicine, and the Vermont

Lung Center in 2005; “Stem Cells and Cell Therapies in Lung Biology and Diseases” sponsored by the NHLBI, Alpha-1 Foundation, ATS, Pulmonary Fibrosis Foundation, University of Vermont College of Medicine, and the Vermont Lung Center in 2007; and “Stem Cells and Cell Therapies in Lung Biology and Diseases” sponsored by the NHLBI, Alpha-1 Foundation, ATS, Emory Center for Respiratory Health, LAM Treatment Alliance, Pulmonary Fibrosis Foundation, University of Vermont College of Medicine, and the Vermont Lung Center in 2009. Those conferences have been instrumental in helping guide research and funding priorities (1–3). Since the 2011 conference, investigations of stem cells and cell therapies in lung biology and diseases have continued to rapidly progress. The field has further expanded to include ex vivo lung bioengineering. A growing number of preclinical studies of immunomodulation and paracrine effects of adult mesenchymal stromal (stem) cells (MSCs) derived from bone marrow, adipose, and other tissues continue to provide evidence of safety and efficacy in animal models of acute lung injury (ALI), asthma, bronchopulmonary dysplasia, chronic obstructive pulmonary disease (COPD), sepsis, silicosis, ventilator-induced lung injury, and other lung diseases. In parallel, more sophisticated understanding of the mechanisms by which MSCs can act has provided growing

insight into their potential applicability for clinical lung diseases. Notably, a pioneering multicenter, double-blinded, randomized placebo-controlled trial of MSCs in patients with moderate to severe COPD has provided valuable safety data for MSC administration to patients with lung diseases and has also suggested potential mechanisms of MSC actions in vivo in patients with lung disease (4). Planned North American investigations of MSC administration in patients with acute respiratory distress syndrome (ARDS), sepsis, and idiopathic pulmonary fibrosis (IPF) are paralleled by an increasing number of clinical investigations of MSCs in lung diseases in other countries. Other cell types, including bone marrow– derived mononuclear cells and human amnion–derived stem cells, also appear to have efficacy in preclinical mouse models of lung diseases and may provide alternative approaches to parallel those using MSCs. Significant advances continue to be made in novel areas of investigation, particularly increasing exploration of three-dimensional culture systems and bioengineering approaches to generate functional lung tissue ex vivo and in vivo. Similarly, progress continues in studies of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with recent data demonstrating more convincing evidence of derivation of cells with phenotypic characteristics of both airway and alveolar epithelial cells. Significant progress also continues

(Received in original form April 19, 2013; accepted in final form May 9, 2013 ) Supported by the Alpha-1 Foundation, American Thoracic Society, LAM Treatment Alliance, Pulmonary Fibrosis Foundation, University of Vermont College of Medicine, University of Vermont Department of Medicine, and the Vermont Lung Center. The conference was also supported in part by National Heart, Lung, and Blood Institute grant R13 HL097533 (D.J.W.). Correspondence and requests for reprints should be addressed to Daniel J. Weiss, M.D., Ph.D., 226 Health Sciences Research Facility, University of Vermont College of Medicine, Burlington, VT 05405. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Ann Am Thorac Soc Vol 10, No 5, pp S25–S44, Oct 2013 Copyright © 2013 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201304-089AW Internet address: www.atsjournals.org

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VERMONT STEM CELL CONFERENCE to be made in investigations of local (endogenous) stem and progenitor cells resident in adult lungs. Advances in lineage tracing approaches and other techniques continue to provide important insights into understanding of the identity and lineage expansion properties of previously identified putative endogenous stem and progenitor populations and suggest an increasingly complex network of cellular repair after injury. Recent data have broadened this beyond consideration of epithelial progenitors to also include endogenous pulmonary vascular and interstitial progenitors. However, ongoing challenges are to better define, access, and manipulate the appropriate niches and to continue to devise more refined lineage tracing and other study mechanisms to define, characterize, and explore potential therapeutic and/or pathologic properties of endogenous lung progenitor cells. This includes studies of lung cancer stem cells, an area of increasing focus and high interest that remains incompletely understood. Another challenge is that most studies of endogenous progenitor cells continue to use mouse models. Correlative information in human lungs remains less well defined. Stem and progenitor cell nomenclature remains a thorny issue, although some progress has been made. Despite suggested guidelines from previous conferences and from other sources, precise definitions and characterizations of specific cell populations, notably the putative endogenous cell populations in the lung as well as mesenchymal stem (stromal) cells and endothelial progenitor cells (EPCs), are not agreed on. In many respects this reflects more sophisticated knowledge about cell plasticity and a realization that phenotypic boundaries are not as rigid as previously believed. Nonetheless, the terms “stem cell” and “progenitor cell” are still used with varying degrees of clarity and precision by different investigators and in recent publications. This continues to complicate comparison of different investigative approaches. A suggested glossary of relevant working definitions applicable to lung, originally presented in the report of the 2007 conference, is again depicted in Table 1. This glossary does not necessarily reflect an overall consensus for the definition of each term and will undergo continuing revision S26

as overall understanding of the cell types and mechanisms involved in lung repair continue to be elucidated. Nonetheless, it remains a useful framework. The conference was divided into five sessions, each featuring a plenary speaker, research talks presented by leading international investigators, and a panel-led debate and discussion. In the first session, “Endogenous Lung Progenitor Cells/Lung Cancer Stem Cells,” after an overview of the field by Wellington Cardoso (Boston University), respective presentations by Emma Rawlins (Cambridge University), Ivan Bertoncello (University of Melbourne), Carla Kim (Boston Children’s Hospital), Susan Reynolds (National Jewish Hospital), and Barry Stripp (Duke University) reviewed the current state of knowledge of endogenous progenitor cell populations, mechanisms regulating their behavior, and their potential to initiate or augment repair. Key points emphasized during this session, as in previous meetings, are that stem cells are operationally defined not solely by their intrinsic developmental potential but by their interaction with the microenvironments in which they reside. Furthermore, the stem cell niche is a dynamic “temporal” niche with the capacity to modify stem cell behavior/ readout in different contexts. Moreover, stem cell–associated markers are not uniquely expressed by stem cells and are unreliable predictors of the “stem” or “progenitor” cell potential of isolated cells. Validation by functional assays and lineage-tracing studies, particularly when interrogating isolated cells where histomorphometric spatial and positional cues are lost, remain increasingly valid and necessary. The session also included a presentation from Mark Magnuson (Vanderbilt University) describing the National Institute of Health’s Beta Cell Consortium and how this type of organizational approach could be applied to the lung endogenous progenitor field. The second session on “Embryonic Stem Cells, iPSCs, and Lung Regeneration” included a featured talk on the current state of knowledge with respect to type 2 alveolar epithelial cells by Jeff Whitsett (Cincinnati Children’s Hospital) followed by presentations from Gustavo Mostoslavsky (Boston

University), Ali Samadikuchaksaraei (Imperial College, London), Darrell Kotton (Boston University), Amy Wong (University of Toronto), Hans-Willem Snoeck (Columbia University), and Brian Davis (University of Texas) highlighting developments in these areas. Notable advances in this area include the improved sophistication in directing both ESCs and iPSCs in vitro through stages involved in generation of definitive endoderm and subsequently into cells with some phenotypic characteristics of airway and alveolar epithelial cells. However, although showing promising progress, full phenotypic and functional characterization of putative lung cells derived from ESCs or iPSCs remains incomplete and is an area in which further exploration is required. The third session, “Bioengineering Approaches to Lung Regeneration,” was expanded to a full day, reflecting the rapid advances in this overall area. The morning session was focused on structure and matrix for three-dimensional scaffolds to be potentially used for ex vivo lung regeneration. A featured overview talk by Dame Julia Polak (Imperial College, London) was followed by presentations from Paolo Macchiarini (Karolinska Institute), Joaquin Cortiella (University of Texas, Galveston), Christine Finck (University of Connecticut), Andrew Hoffman (Tufts University), Peter Lelkes (Temple University), Angela PanoskaltsisMortari (University of Minnesota), and Zachary Borg (University of Vermont) exploring advances in scaffold systems, particularly with respect to whole lung decellularization and recellularization. The afternoon session was focused on functional aspects of ex vivo lung bioengineering and included presentations by Doris Taylor (University of Minnesota), David Hoganson (Washington University), Harald Ott (Harvard University), Laura Niklason (Yale University), Daniel Huh (Harvard University), and Daniel Tschumperlin (Harvard University). A final presentation on imaging approaches for lung bioengineering was presented by Jason Woods (Washington University). The fifth session, “EPCs, MSCs, and Cell Therapy Approaches for Lung Diseases,” highlighted recent advances in preclinical and clinical cell therapy approaches

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VERMONT STEM CELL CONFERENCE Table 1. Glossary and definition of terminology Potency: Sum of developmental options available to cell Totipotent: Ability of a single cell to divide and produce all the differentiated cells in an organism, including extraembryonic tissues, and thus to (re)generate an organism in total. In mammals only the zygote and the first cleavage blastomeres are totipotent. Pluripotent: Ability of a single cell to produce differentiated cell types representing all three embryonic germ layers and thus to form all lineages of a mature organism. Example: embryonic stem cells Multipotent: Ability of adult stem cells to form multiple cell types of one lineage. Example: hematopoietic stem cells Unipotent: Cells form one cell type. Example: spermatogonial stem cells (can only generate sperm) Reprogramming: Change in epigenetics that can lead to an increase in potency, dedifferentiation. Can be induced by nuclear transfer, cell fusion, genetic manipulation Transdifferentiation: The capacity of a differentiated somatic cell to acquire the phenotype of a differentiated cell of the same or different lineage. An example is epithelial–mesenchymal transition (EMT), a process whereby fully differentiated epithelial cells undergo transition to a mesenchymal phenotype giving rise to fibroblasts and myofibroblasts. Plasticity: Hypothesis that somatic stem cells have broadened potency and can generate cells of other lineages, a concept that is controversial in mammals. Embryonic stem cell (ESC): Cell lines developed from the inner cell mass of early developing blastocysts. ESCs have the capacity for selfrenewal and are pluripotent, having the ability to differentiate into cells of all embryologic lineages and all adult cell types. However, ESCs cannot form extraembryonic tissue, such as trophectoderm. Adult stem cell: Cells isolated from adult tissues, including bone marrow, adipose tissue, nervous tissue, skin, umbilical cord blood, and placenta, that have the capacity for self-renewal. In general, adult stem cells are multipotent, having the capacity to differentiate into mature cell types of the parent tissue. Some populations of adult stem cells, such as MSCs, exhibit a range of lineage differentiation that is not limited to a single tissue type. Whether adult stem cells exhibit plasticity and can differentiate into a wider variety of differentiated cells and tissues remains controversial. Adult tissue-specific stem cell: Same as adult stem cells but with defined tissue specificity. A relatively undifferentiated cell within a given tissue that has the capacity for self-renewal through stable maintenance within a stem cell niche. Adult tissue-specific (endogenous) stem cells have a differentiation potential equivalent to the cellular diversity of the tissue in which they reside. The hematopoietic stem cell is a prototypical adult tissue stem cell. Induced pluripotent stem cell (iPSC): Reprogrammed adult somatic cells that have undergone dedifferentiation after the expression of reprogramming transcription factors such as Oct 3/4, Sox2, c-Myc, and Klf4. iPSCs are similar to ESCs in morphology, proliferation, gene expression, and ability to form teratomas. In vivo implantation of iPSCs results in formation of tissues from all three embryonic germ layers. iPSCs have been generated from both mouse and human cells. Progenitor cell: A collective term used to describe any proliferative cell that has the capacity to differentiate into different cell lineages within a given tissue. Unlike stem cells, progenitor cells have limited or no self-renewal capacity. The term progenitor cell is commonly used to indicate a cell can expand rapidly but undergoes senescence after multiple cell doublings. Terminology that takes into account the functional distinctions among progenitor cells is suggested below. Transit-amplifying cell: The progeny of a endogenous tissue stem cell that retain relatively undifferentiated character, although more differentiated than the parent stem cell, and have a finite capacity for proliferation. The sole function of transit-amplifying cells is generation of a sufficient number of specialized progeny for tissue maintenance. Obligate progenitor cell: A cell that loses its ability to proliferate once it commits to a differentiation pathway. Intestinal transit-amplifying cells are obligate progenitor cells. Facultative progenitor cell: A cell that exhibits differentiated features when in the quiescent state yet has the capacity to proliferate for normal tissue maintenance and in response to injury. Bronchiolar Club cells are an example of this cell type. Classical stem cell hierarchy: A stem cell hierarchy in which the adult tissue stem cell actively participates in normal tissue maintenance and gives rise to a transit-amplifying cell. Within this type of hierarchy, renewal potential resides in cells at the top of the hierarchy (i.e., the stem and transit-amplifying cell), and cells at each successive stage of proliferation become progressively more differentiated. Nonclassical stem cell hierarchy: A stem cell hierarchy in which the adult tissue stem cell does not typically participate in normal tissue maintenance but can be activated to participate in repair after progenitor cell depletion. Rapidly renewing tissue. Tissue in which homeostasis is dependent on maintenance of an active mitotic compartment. Rapid turnover of differentiated cell types requires continuous proliferation of stem and/or transit-amplifying cells. A prototypical rapidly renewing tissue is the intestinal epithelium. Slowly renewing tissue: Tissues in which the steady-state mitotic index is low. Specialized cell types are broadly distributed, long-lived, and a subset of these cells, the facultative progenitor cells, retain the ability to enter the cell cycle. The relative stability of the differentiated cell pool is paralleled by infrequent proliferation of stem and/or transit-amplifying cells. The lung is an example of a slowly renewing tissue. Hematopoietic stem cell: Cell that has the capacity for self-renewal and whose progeny differentiate into all of the different blood cell lineages, including mature leukocytes, erythrocytes, and platelets. Endothelial progenitor cell (EPC): Circulating cells that have the potential to proliferate and differentiate into mature endothelial cells. Studies of EPCs have been complicated by the use of the same terminology to define at least two different cell populations that have different cell surface markers, different cell sources, and different abilities to differentiate into mature endothelial cells in vitro and in vivo. There is a critical need to develop a consensus definition of EPCs with particular emphasis on the functional capabilities of these cells. Mesenchymal stromal (stem) cell (MSC): Cells of stromal origin that can self-renew and have the ability to differentiate into a variety of cell lineages. Initially described in a population of bone marrow stromal cells, they were first described as fibroblastic colony-forming units, subsequently as marrow stromal cells, then as mesenchymal stem cells, and most recently as multipotent mesenchymal stromal cells or MSCs. MSCs have now been isolated from a wide variety of tissues, including umbilical cord blood, Wharton’s jelly, placenta, adipose tissue, and lung. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) has recently published the minimal criteria for defining (human) MSCs. MSCs have been described to differentiate into a variety of mature cells types and may also have immunomodulatory properties. (Continued )

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VERMONT STEM CELL CONFERENCE Table 1. (CONTINUED ) Fibrocyte: A cell in the subset of circulating leukocytes that produce collagen and home to sites of inflammation. The identity and phenotypic characterization of circulating fibrocytes is more firmly established than that for EPCs. These cells express the cell surface markers CD34, CD45, CD13, and MHC II and also express type 1 collagen and fibronectin. Bronchiolar stem cell: A term applied to a rare population of toxin (i.e., naphthalene)-resistant Club cell secretory protein (CCSP)-expressing cells that localize to neuroepithelial bodies and the bronchoalveolar duct junction of the rodent lung. These cells proliferate infrequently in the steady state but increase their proliferative rate after depletion of transit-amplifying (Club) cells. Lineage-tracing studies indicate that these cells have the differentiation potential to replenish specialized cell types of the bronchiolar epithelium. Human correlates have not yet been identified. Bronchioalveolar stem cell: A term applied to a small population of cells located at the bronchoalveolar duct junction in mice identified in vivo by dual labeling with CCSP and surfactant protein C (SP-C) and by resistance to destruction with toxins (i.e., naphthalene). In culture, some of the dual-labeled cells also express Sca1 and CD34, self-renew, and give rise to progeny that express either CCSP, pro–SP-C, or aquaporin 5, leading to speculation that a single cell type has the capacity to differentiate into both bronchiolar (Club cells) and alveolar (type 1 and 2 pneumocytes) lineages. At present, the relationship of the cells studied in vitro to those observed by dual labeling in vivo is unclear. Human correlates have not yet been identified. Modified by permission from Reference 3.

for lung diseases. After a featured presentation by Darwin Prockop (Texas A&M), talks given by Jacques Galipeau (Emory University), Donald Phinney (Scripps Institute, Florida), Michael Matthay (UCSF), Sam Janes (University College, London), Meagan Goodwin and Daniel Weiss (University of Vermont), Mervin Yoder (Indiana University), and Duncan Stewart (University of Ottawa) highlighted different areas of advances in EPC and MSC biology and further progress toward an expanded investigation of clinical cell therapy approaches for different lung diseases, including lung cancers. The final session, “Summation and Direction,” featured a review and open discussion of current issues led by a panel featuring representatives of the NHLBI, U.S. Food and Drug Administration (FDA), and each of the sponsoring nonprofit respiratory disease organizations. The panel consisted of Carol Blaisdell (NHLBI), Amy Farber (Founder and CEO, LAM Treatment Alliance), Donald Fink Jr. (FDA), Christine Kelley (National Institute of Bioimaging and Biomedical Engineering), Michael Matthay (on behalf of the ATS), Neal Pellis, Ph.D. (National Aeronautics and Space Adminstration), Daniel Rose (CEO, Pulmonary Fibrosis Foundation), and Adam Wanner (Alpha-1 Foundation). The conference concluded with vigorous discussion on future research and funding priorities, led by Darwin Prockop (Texas A&M). As in previous conferences, discussion was spirited in all areas. It was agreed that strong emphasis must continue be placed on animal models of human lung diseases, with a focus on studies that incorporate relevant functional S28

outcome measures. Nonetheless, the safety results obtained with the trial of MSCs in COPD and the increasing body of data demonstrating efficacy in other inflammatory and immune-mediated lung injury disease models suggest a potential role in inflammatory and immunemediated lung diseases even in the absence of a comprehensive understanding of the mechanisms by which the MSCs are acting. It was acknowledged by all participants that ex vivo lung bioengineering and further investigation into the role of endogenous lung progenitor cells remain timely and exciting areas of study. Nonetheless, there are many areas in which our understanding of the processes and mechanisms remain poorly understood. Recommendations for areas of continued and future investigation are presented in Table 2. More extensive details on each session are presented below. The conference program, executive summaries for each speaker, and abstracts from the poster sessions are included in the online supplement. In addition, a companion article provides a comprehensive review of the relevant literature spanning the period between the 2011 workshop through December 2012. The combined references for both this conference report and from the comprehensive review are found after the review. In accordance with recent guidelines from the ATS and other Respiratory Disease organizations, the terms “Clara cell” and “Clara cell secretory protein” have been replaced by the terms “Club cell” and “Club cell secretory protein (CCSP),” respectively (5, 6).

Detailed Session Summaries Session 1: Endogenous Lung Progenitor and Lung Cancer Stem Cells

The endogenous lung progenitor cell and lung cancer stem cell session at this year’s conference highlighted two complementary approaches to studying lung epithelial progenitor cells. The first approach revolved around the discipline of developmental biology and emphasized the importance of identifying new markers of potentially very heterogeneous populations of progenitor cells that occur at distinct positions along the respiratory tree. This approach relies on the precise identification of unique markers of different progenitor cells and, in turn, permits the creation of murine genetic models to lineage trace the fate of any one particular progenitor cell type identified by such a unique marker in an injury of one’s choice. The creation of such lineage-tracing mice also permits the sorting, purification, and ultimately genetic modulation of these progenitor cells in murine disease models. The second approach borrows from the remarkable advances made in hematopoietic stem cell biology and relies on the use of multiparameter fluorescenceactivated cell sorter (FACS) sorting and functional colony-forming cell assays to identify and prospectively isolate increasingly enriched populations of candidate progenitor cells, which can be hierarchically ordered on the basis of their proliferative and differentiation potential and growth factor requirements in culture. This second approach has the advantage that it can be applied to human systems as easily as it can be to the mouse. A take-

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VERMONT STEM CELL CONFERENCE Table 2. Overall conference summary recommendations Basic For studies evaluating putative engraftment, advanced histologic imaging techniques (e.g., confocal microscopy, deconvolution microscopy, electron microscopy, laser capture dissection, etc.) must be used to avoid being misled by inadequate photomicroscopy and immunohistochemical approaches. Imaging techniques must be used in combination with appropriate statistical and other analyses to maximize detection of rare events. Continue to elucidate mechanisms of recruitment, mobilization, and homing of circulating or therapeutically administered cells to lung epithelial, interstitial, and pulmonary vascular compartments for purposes of either engraftment or immunomodulation. Encourage new research to elucidate molecular programs for development of lung cell phenotypes. Continue to refine the nomenclature used in study of endogenous and exogenous lung stem cells. Comparatively identify and study endogenous stem/progenitor cell populations between different lung compartments and between species. Increase focus on study of endogenous pulmonary vascular and interstitial progenitor populations. Develop robust and consistent methodologies for the study of endogenous lung stem and progenitor cell populations. Develop more sophisticated tools to identify, mimic, and study ex vivo the relevant microenvironments (niches) for study of endogenous lung progenitor/stem cells. Continue to develop functional outcome assessments for endogenous progenitor/stem cells. Elucidate how endogenous lung stem and progenitor cells are regulated in normal development and in diseases. Identify and characterize putative lung cancer stem cells and regulatory mechanisms guiding their behavior. Continue to elucidate mechanisms by which embryonic and induced pluripotent stem cells develop into lung cells/tissue. Develop disease-specific populations of ESCs and iPSCs, for example for CF and a1-antitrypsin deficiency, with the recognition that no strategy has yet been devised to overcome the propensity of ESCs and iPSCs to produce tumors. Continue to explore lung tissue bioengineering approaches, such as artificial matrices and three-dimensional culture systems, for generating lung ex vivo and in vivo from stem cells, including systems that facilitate vascular development. Evaluate effect of environmental influences, including oxygen tension, and mechanical forces, including stretch and compression pressure, on development of lung from stem and progenitor cells. Identify additional cell surface markers that characterize lung cell populations for use in visualization and sorting techniques. Strong focus must be placed on understanding immunomodulatory and other mechanisms of cell therapy approaches in different specific preclinical lung disease models. Improved preclinical models of lung diseases are necessary. Disseminate information about and encourage use of existing core services, facilities, and web links. Actively foster interinstitutional, multidisciplinary research collaborations and consortiums as well as clinical/basic partnerships. Include a program of education on lung diseases and stem cell biology. A partial list includes NHLBI Production Assistance for Cellular Therapies (PACT), NCRR stem cell facilities, GMP Vector Cores, small animal mechanics, and CT scanner facilities at several pulmonary centers. Translational Support high-quality translational studies focused on cell-based therapy for human lung diseases. Preclinical models will provide proof of concept; however, these must be relevant to the corresponding human lung disease. Disease-specific models, including large animal models where feasible, should be used and/or developed for lung diseases. Basic/translational/preclinical studies should include rigorous comparisons of different cell preparations with respect to both outcome and toxicological/safety endpoints. For example, it is not clear which MSC or EPC preparation (species and tissue source, laboratory source, processing, route of administration, dosing, vehicle, etc.) is optimal for clinical trials in different lung diseases Incorporate rigorous techniques to unambiguously identify outcome measures in cell therapy studies. Preclinical models require clinically relevant functional outcome measures (e.g., pulmonary physiology/mechanics, electrophysiology, and other techniques). Clinical Proceed with design and implementation of initial exploratory safety investigations in patients with lung diseases where appropriate, such as ARDS/ALI, asthma, and others. This includes full consideration of ethical issues involved, particularly which patients should be initially studied. Provide increased clinical support for cell therapy trials in lung diseases. This includes infrastructure, use of NIH resources such as the PACT program, and the NCRR/NIH Center for Preparation and Distribution of Adult Stem Cells (MSCs; http://medicine.tamhsc.edu/irm/ msc-distribution.html), coordination among multiple centers, and registry approaches to coordinate smaller clinical investigations. Clinical trials must include evaluations of potential mechanisms, and this should include mechanistic studies as well as assessments of functional and safety outcomes. Trials should include, whenever feasible, collection of biologic materials, such as lung tissue, BAL fluid, blood, etc., for investigation of mechanisms as well as for toxicology and other safety endpoints. Partner with existing networks, such as ARDSNet or ACRC, nonprofit respiratory disease foundations, and/or industry as appropriate to maximize the scientific and clinical aspects of clinical investigations. Integrate with other ongoing or planned clinical trials in other disciplines in which relevant pulmonary information may be obtained. For example, inclusion of pulmonary function testing in trials of MSCs in graft vs. host disease will provide novel and invaluable information about potential MSC effects on development and the clinical course of bronchiolitis obliterans. Work with industry to have access to information from relevant clinical trials. Definition of abbreviations: ACRC = Asthma Clinical Research Center; ALI = acute lung injury; ARDS = acute respiratory distress syndrome; BAL = bronchoalveolar lavage; CF = cystic fibrosis; ESC = embryonic stem cell; GMP = good manufacturing process; iPSC = induced pluripotent stem cell; MSC = mesenchymal stromal (stem) cell; NCRR = National Center for Research Resources; NHLBI = National Heart, Lung, and Blood Institute; NIH = National Institutes of Health.

home message of the conference was that there needs to be a confluence of these two modalities of study in the next decade, Vermont Stem Cell Conference

mirroring what has been accomplished in the field of hematopoiesis. For now, the major barriers to progress include the lack

of definitive markers to segregate progenitor cells at different levels of the airway tree, whether by FACS or genetic S29

VERMONT STEM CELL CONFERENCE approaches, and most importantly the availability of informative gold-standard functional in vivo assays for stem cells akin to bone marrow transplantation to demonstrate the true regenerative potential of any purified cell population from mouse or human. Wellington Cardoso opened the section with a thoughtful review of the state of the field in lung progenitor cell biology, arguing that it is has been a challenging task to define the properties of lung progenitor cells due to the great structural complexity and diversity of the cell types and the slow turnover of the respiratory epithelium under homeostatic conditions. He noted that most studies therefore focus on candidate endogenous progenitor or stem cells using models of adult lung injury and repair (7). Although these models provide important insights into the pathways and cellular activities involved in the lung’s regenerative response, they may not necessarily reflect the behavior of the endogenous progenitors in the uninjured lung (8). Based on lineage tracing and injury-repair models, three different cell types have been accepted classically as endogenous progenitors of the adult respiratory system: basal and Club cells in airways and type II cells in the alveoli. There is evidence that these cell types actually encompass distinct subpopulations with different features and functions. Club cells, for example, are a highly heterogeneous population of facultative progenitors in intrapulmonary airways in rodents and humans, being able to self-renew and give rise to ciliated cells. This heterogeneity is manifested in multiple ways, such as in their ability to undergo mucous metaplasia (9) or survive naphthalene injury and mount a regenerative response (10). These properties are associated with the regional distribution of these cells and seem to depend on interactions with the microenvironment, through mechanisms still largely obscure. Studies to further explore heterogeneity of basal, Club, and type II cells and their precursors are needed and will be of great value. Dr. Cardoso also noted that new candidate adult lung progenitors have been recently reported. A population of a6b41 cells present in the normal adult mouse lung at the bronchioalveolar duct junction and alveoli has been shown to possess the S30

capacity for proliferation and further differentiation ex vivo. When mixed with embryonic lung cells in serial dilutions, they form well-defined airway and saccular structures in kidney capsule assays. Moreover, they participate in the regenerative response of the lung epithelium post–bleomycin injury. A novel population of progenitor cells has been also described in submucosal glands of the murine trachea. These cells survive hypoxic-ischemic injury and play a role in repair of the submucosal glands (11). Additionally, a single CD49f(bright)/Sca1 (1)/ALDH(1) basal cell has been reported to generate rare label-retaining cells and abundant label-diluting cells. The property of self-renewal of these cells was tested by serial passage at clonal density and analysis of clone-forming cell frequency. A single clone could be passaged up to five times (12). Dr. Cardoso also noted that recent claims of a human lung stem cell cKit1 capable of regenerating all elements of the injured mouse lung have been controversial (13). Dr. Cardoso finally highlighted the Notch pathway as crucial actor in cell fate choice in differentiating airway progenitors during development or regeneration. Murine models show that disruption of Notch signaling alters dramatically the balance of ciliated and secretory cells (14–16). Alterations in the Notch pathway have been associated with aberrant programs of differentiation in human lung conditions, including COPD and cancer (17, 18). Notch is likely to mediate the responses of the endogenous progenitors to the microenvironment critical to control cell fate. Although differences exist between human and animal models, the characterization and in-depth knowledge of developmental signaling in different models will further the current understanding of endogenous adult progenitors in the lung. Dr. Emma Rawlins then described her work in embryonic lung development. Her group has shown that during lung branching morphogenesis the distal-most ID21 epithelial cells are a multipotent progenitor population. These cells both self-renew during development and give rise to all of the different lung epithelial cell types (19). However, these multipotent embryonic progenitors are not maintained after birth. By contrast, in the adult lung, multiple progenitor cell populations of

more restricted potency are necessary for maintenance and repair of the epithelium (20). In distinction to the molecular mechanisms governing embryonic progenitor cell behavior, the mechanisms governing these adult progenitor cell populations remain largely obscure. Dr. Rawlins noted that adult lung stem cell biology can build on the data that have been collected by lung developmental biologists. Indeed, many of the same molecular mechanisms governing both embryonic and adult lung progenitor cells and embryonic gene regulation networks can reappear in the adult lung pathologies, including cancer (21). Finally, it was pointed out that knowledge of the mechanisms that control embryonic lung development will provide a framework for driving the differentiation of iPSCs into lung progenitors (22). Ivan Bertoncello then reviewed the significant progress made in recent years in characterizing and analyzing the proliferative potential and organization of candidate epithelial stem/progenitor cells in the adult lung using FACS-based cell sorting approaches and in vitro colonyforming assays. The Bertoncello laboratory has described a rare population of EpCAMposSca-1lowa6posb4posCD24low lung epithelial stem/progenitor cells, which give rise to airway or alveolar lineagerestricted colonies as well as colonies of mixed lung epithelial cell lineages when cocultured in a three-dimensional matrigel assay together with supporting mesenchymal stromal cells and cytokines (23). Parallel studies, and the refinement of cell separative strategies used by other laboratories, have since shown that candidate stem/progenitor cells isolated from different regions along the proximodistal lung axis share a similar if not identical biomarker repertoire. These include bronchioalveolar stem cells (BASCs) (24), bronchiolar progenitor cells (25), and multipotent a6posb4pos cells, recently shown to recapitulate lung morphogenesis in a sub-kidney capsule lung organoid transplant assay (26). Dr. Bertoncello argued that although this reductionist approach provides powerful tools to monitor the status of stem/ progenitor pools in the normal and diseased lung, and to identify regulatory factors, cytokines, and pathways that specify their fate, much still remains to be done to precisely understand what these

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VERMONT STEM CELL CONFERENCE assays are telling us about the organization, regulation, and regenerative potential of endogenous lung stem cells in situ. In doing so, he identified a number of questions and gaps in our knowledge about the properties of candidate stem/progenitor cells that need to be resolved to assess the usefulness of these surrogate assays in identifying targets around which cellular therapies for lung disease can be tailored. For example, current protocols have enabled us to prospectively isolate cell fractions highly enriched for colonyforming cells but have not enabled us to resolve colony-forming cells of differing developmental potential. This is consistent with the premise that stem and progenitor cells are characterized by the expression of multiple markers that collectively specify the stem cell state but are not unique to individual stem/progenitor cell types. But, it is unclear whether the readout of index assays is indicative of cofractionation of heterogeneous progenitor cells expressing common biomarkers or the stochastic commitment of homogeneous progenitor cells to different fates in response to different microenvironmental cues. Dr. Bertoncello also reminded the audience that the regenerative capacity of stem and progenitor cells in vivo is determined not solely by their intrinsic developmental potential but also by the reciprocal signaling between stem/ progenitor cells and their niche microenvironment, which includes stromal and extracellular matrix (ECM) elements, accessory cells, as well as soluble and insoluble factors that also respond to insult or injury, temporally and dynamically altering the regulatory properties of the stem cell niche (27, 28). Consequently, in vitro clonogenic assays are powerful but imperfect tools for modeling stem/ progenitor cell hierarchies, and there is a pressing need for the development of “gold standard” in vivo assays akin to hematopoietic stem cell transplant assays to establish the precise relationship between candidate stem/progenitor cells identified in vitro and regenerative cells in vivo. He concluded by raising the issue that targeting the dysregulated lung epithelial/progenitor cell niche may prove as important as targeting epithelial stem/progenitor cells in developing cell-based therapies in lung regenerative medicine (29). Dr. Barry Stripp kept up with this theme and argued that there are regionally Vermont Stem Cell Conference

distinct regenerative zones that function in concert to maintain and repair the epithelium of postnatal conducting airways (25). The Stripp laboratory has used lineage tracing coupled with cell fractionation approaches to understand how progenitors from different airway regions behave in a defined culture environment. His laboratory’s data suggest that the positional identity of regionally distinct airway progenitor cells is controlled through intrinsic mechanisms. Gene expression profiling was used to define candidate molecular markers that distinguish regional progenitor cells and provide new tools for lineage tracing and functional analysis in vivo. Parallel studies performed using human lung tissue suggest that principles developed through use of mouse models are broadly applicable to other mammalian species, including humans. Dr. Susan Reynolds then focused on a specific subset of basal epithelial progenitor cells, the facultative basal progenitor (FBP), and roles for b-catenin in regulation of FBP proliferation and differentiation (30). Using tracheal air– liquid interface cultures, Reynolds showed that these cultures could be divided into two phases: basal cell proliferation, which results in expansion of the basal cell population, and basal cell differentiation into ciliated or Club-like cells. Using genetic stabilization of b-catenin in basal cells, her laboratory demonstrated that b-catenin–dependent signaling had a minor impact on the level of proliferation that resulted in population expansion. However, stabilized b-catenin shortened the post-confluence period of basal cell proliferation. She speculated that this effect was due to initiation of basal cell differentiation. Stabilization of b-catenin enhanced basal-to-ciliated cell differentiation twofold but completely blocked basal-to– Club-like cell differentiation. These results argued that b-catenin regulated basal cell fate by favoring the generation of ciliated cells over Club cells. A loss-of-function allele was also used to demonstrate that b-catenin was necessary for early but not late basal-tociliated cell differentiation and basal-to– Club-like cell differentiation as well as basal cell proliferation and long-term survival of basal cells. Finally, Dr. Carla Kim described her initial isolation of BASCs via isolation of Sca1-positive, CD31/CD45-negative cells by FACS and her demonstration that these cells

can self-renew on mouse embryonic fibroblast feeder layers or differentiate on a two-dimensional Matrigel setting (31). The Kim Lab has more recently developed a means to better enrich for BASC by FACS sorting CD31/45-negative, Sca1-low, CD24-low, Epcam-positive cells, offering an improved method of isolating putative lung stem cells, which is in good agreement with others’ marker profile for lung stem cells (work of Stripp and Bertoncello laboratories). Dr. Kim then referenced that the Hogan Laboratory has recently shown with CCSP-CreERT2;LSL-YFP mice lineage-tracing strategies that CCSP lineage-marked cells do not contribute to alveolar epithelia during development, suggesting that BASCs do not play an important role in maintaining alveolar cell types during steady-state homeostatic conditions. Additionally, she referenced these studies demonstrating that CCSPpositive cells cannot give rise to alveolar lineages after treatment with hyperoxia. She verified that her group does not observe a BASC response to hyperoxia treatment. However, she hypothesized that BASCs play a key role in the response of adult lungs to particular contexts of bronchiolar and alveolar injury, such as the naphthalene and bleomycin response. To further test this hypothesis, her group used CCSPCreER;LSL-YFP mice and performed lineage tracing studies with bleomycin treatment. Importantly and in contrast to the Hogan Laboratory hyperoxia results, Kim has determined that there is a significance increase in CCSP lineagetagged cells in the alveolar space after bleomycin treatment (Kim Lab, unpublished). This is consistent with her hypothesis that BASCs are capable of differentiation to alveolar epithelia in vivo in particular contexts. Alternatively, the data may also indicate Club cells have this capacity. To distinguish between these possibilities, it will be necessary to achieve BASC-specific genetic marking in vivo. Dr. Kim also described her identification of an important new role in adult cells for Bmi1in the repression of genes that are typically regulated by imprinting mechanisms (24). Expression of p57, Igf2, and other imprinted genes was higher in BASCs compared with AT2 cells, was required for wild-type lung stem cell self-renewal in culture, and correlated with the ability of stem cells to S31

VERMONT STEM CELL CONFERENCE effectively repair damaged lung epithelium in vivo. Session 2: Embryonic Stem Cells, iPSCs, and Lung Regeneration

This session was focused on the development of induced pluripotent stem cell lines from normal donors and patients with disease to serve as a renewable source of cells. In addition to the widely anticipated use of these cells for transplantation or regenerative medicine applications, it was clear that a more immediate impact of iPSC technology is the generation of respiratory and other lineage cells from normal donors and patients with disease to study in vitro. Examples were provided for the use of iPSC-derived respiratory cells to study early human development, including germ layer and cell lineage commitment, and differentiation. Other studies generated iPSC lines from patients with disease to study cellular mechanisms of disease, evaluate gene therapies, and screen drugs, as outlined below for each presentation. In the first presentation in this session, Dr. Jeffrey Whitsett (Cincinnati Children’s Research Foundation) provided an overview of the large number of respiratory disorders that could potentially benefit from the development of respiratory cellular repair and regenerative medicine applications in the lung, including bronchopulmonary dysplasia (BPD), COPD, ARDS, cystic fibrosis (CF), and others. He highlighted the complex structure and physiology of the lung at the organ and cellular levels, which need to be considered and understood before undertaking cell-based regenerative therapies in the lung. Specifically, cellular therapies for lung disease would need to ensure repair or normalization of surfactant homeostasis, alveolar–capillary integrity, mucociliary clearance, host defense, normal cell proliferation, differentiation, and repair processes. He then used alveolar homeostasis as an example of the high level of cellular organization and regulation required to produce and maintain normal tissue or repair damaged tissue. He provided many examples of disease models in which an imbalance in the spatial and temporal, proliferation or differentiation, or cellular interactions of cells in the alveolus can lead to injured or dysfunctional tissue. This presentation highlighted the need for potential cellular S32

therapies to produce a tightly controlled regulation of multiple cell proliferation and differentiation and the establishment of cell contact with other lineages in the alveolus to repair lung tissue, without introducing serious consequences. The next presentation by Dr. Gustavo Mostoslavsky (Boston University) provided an overview of the methodologies for reprogramming and generating induced pluripotent stem cells, including a review of the rapid pace of innovations in reprogramming. He presented an example of reprogramming and iPSC line generation and testing using a single excisable lentiviral vector (STEMCCA), developed in his laboratory, that reprogrammed fibroblasts and other cells with a high efficiency (32). The expression cassette can be removed after a transient expression of cre recombinase to produce iPSCs without the transgene cassette. He also showed transcriptional analysis demonstrating that cells that had the transgene cassette removed were very similar to embryonic stem cells. He provided an overview of the developmental differentiation approach in which in vitro differentiation protocols are developed to match the conditions and factors that regulate embryonic development for specific embryonic germ layers and tissues. For example, definitive endoderm can be induced by addition of activin A, and further differentiation into specific lineages can be induced by altering the environment, such as the addition of fibroblast growth factor 10 (FGF10) to induce lung differentiation. He then discussed studies from his laboratory in which iPSC lines from patients with many genetic diseases have been generated, including CF and a1-antitrypsin deficiency, and highlighted the power of using these iPSC lines to model disease. He concluded his presentation with a comment that despite the rapid pace of iPSC research, clinical application of these cells is many years away, and many studies will be required to determine the safety of transplanting iPSC-derived cells. He cautioned that the “stem cell tourism,” whereby patients are receiving experimental therapies in uncontrolled and unregulated environments, has the potential to hurt patients receiving the stem cells and creates risks for the field of stem cell research to lose credibility with the public after potential medical disasters

Dr. Ali Samadikuchaksaraei (Imperial College of London) spoke about his strategy of optimizing the production of alveolar epithelial cells from mouse and human ESCs. He reviewed his work optimizing many factors in the culture system, including growth factors, conditioned medium and cell lysates from different respiratory cell types and populations, and embryonic lungs (33, 34). He then focused on his development of a bioreactor system to scale-up and simplify the production of respiratory cells by encapsulation of embryoid bodies, culture in rotating bioreactors, and addition of conditioned media, which produced alveolar type I and II cells that could be maintained for 100 days. He outlined his current focus of optimizing differentiation by building an in vitro respiratory unit system through the addition of other respiratory cell populations, such as epithelial, mesenchymal, and endothelial cells, as well as optimizing other culture parameters, including nutrition, pH, and oxygen tension. Dr. Darrell Kotton (Boston University) presented his studies using a developmental approach to generate respiratory cells from mouse ESCs. This approach uses a stepwise differentiation of ESCs and iPSCs through definitive endoderm to foregut endodermal progenitors and subsequently to respiratory progenitors. The first stage of this process is efficient, with greater than 80% of the cells effectively differentiated toward definitive endoderm using activin A in embryoid bodies. His studies focused on optimizing the second phase of differentiation from definitive endoderm to respiratory progenitors through the use of a mouse ESC reporter line in which green fluorescent protein was knocked into the Nkx2.1 locus. In this study, differentiation conditions that promote respiratory progenitor differentiation could be readily screened by fluorescence microscopy or flow cytometry, and the conditions supporting optimal differentiation could readily be identified through the expression of green fluorescent protein. They used this system to determine that inhibition of bone morphogenic protein (BMP) and transforming growth factor (TGF)-b signaling reduced “posterior” patterning and led to increased ventralization of foregut endoderm, resulting in an increase of NKX2.1 endodermal progenitors to 20%. These Nkx2.1 progenitors were similar to

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VERMONT STEM CELL CONFERENCE primordial lung progenitors by gene expression profile. These progenitors could then be induced to respiratory differentiation, as evident from the expression of a wide variety of respiratory epithelial markers after further differentiation in FGF2 and FGF10. Future studies will work on the later differentiation events in fetal lung development to identify the signals regulating the development and contribution of the primordial lung progenitors to continued respiratory development (35). Dr. Amy Wong (The Hospital for Sick Children) presented studies on the development of iPSCs from patients with CF to serve as cellular models of disease, drug discovery, and regenerative medicine. She described the development of a step-wise differentiation system that mimics lung development to generate human airway epithelium to test drugs for CF. The differentiation system used carefully timed addition of morphogens to direct differentiation through definitive endoderm and anterior ventral foregut endoderm, embryonic lung progenitor, and immature lung growth. The cells were then cultured at air–liquid interface to generate a heterogeneous, polarized, and functional airway epithelium. These cultures developed dome-like structures with an adsorptive epithelium capable of ion and fluid transport. The epithelium that formed was similar to large airways with expression of genes consistent with basal cells (p63), goblet cells (MUC5), and ciliated cells (FOXJ1). The Cystic Fibrosis Transmembrane Regulator (CFTR) was localized to the apical membrane, as in the native airways. They next developed iPSC lines from patients with the d508 CFTR mutation from patients with CF (36). Dr. Wong demonstrated that these lines were pluripotent and could generate a functional epithelial layer. These differentiated airway epithelial cultures were then used to screen chemical compounds capable of stabilizing the mutant CFTR at the apical membrane and identified one candidate drug. Ongoing studies will evaluate whether this stabilization and localization of CFTR also resulted in functional ion channels. Future studies will use these iPSC-derived epithelial layers to screen and identify novel CFTR correctors and potentiators. Dr. Hans-Willem Snoeck (Mount Sinai School of Medicine) focused on improving the generation of anterior foregut endoderm from human pluripotent stem Vermont Stem Cell Conference

cells. He highlighted that there are many studies demonstrating the efficient generation of hindgut (intestine), mid-gut (pancreatic endoderm), and posterior foregut endoderm (hepatocytes). However, the generation of anterior foregut lineages has been more challenging. He showed that this is due in part to a mid/posterior bias of activin A–induced endoderm. He screened for signals to enrich anterior endoderm formation in human ESC differentiation and reduce ectoderm and mesoderm differentiation. His studies demonstrated that dual inhibition of TGFb and BMP signaling after the formation of activin A–induced endoderm resulted in a highly enriched population of anterior foregut endoderm. Subsequent addition of Wnt3a, keratinocyte growth factor (KGF), FGF10, epidermal growth factor, and BMP4 to the culture induced cellular lineage differentiation toward an anterior ventral foregut phenotype (22). Subsequent screening of 400 culture conditions on the anterior foregut endoderm identified an optimal combination for the production of alveolar epithelial type II cells. The optimal conditions included continued Wnt, FGF10, and KGF addition to the cultures from Days 10 to 19. This study demonstrated that optimal culture requires highly spatial-temporal addition of factors to mimic embryonic development and achieve optimal differentiation into respiratory epithelium. Dr. Brian Davis (University of Texas Health Science Center) presented his studies on the generation of iPSCs from patients with the inherited respiratory diseases surfactant B deficiency and CF as well as gene correction of the mutant genes. He presented studies in which he generated an iPSC line from a dI507/dF508 CFTR compound heterozygote. The iPSCs were then subject to zinc finger nuclease (ZFN)mediated gene correction that used a donor construct that corrected the CFTR gene and introduced a puro-tk selection cassette into the adjacent intron 10. After transfection, the iPSCs were selected with puromycin and molecularly screened to identify clones that had undergone ZFN editing to correct the CFTR genes. They also demonstrated that the corrected cells expressed the wild-type CFTR after differentiation into an airway epithelial layer. He next presented their studies developing iPSCs from patients with surfactant protein B deficiency with ongoing

studies performing ZFN gene correction of the surfactant protein B mutation. Future studies will include generation of alveolar type II epithelial cells and evaluation of surfactant production in the gene-corrected cells. In summary, this session highlighted that embryonic stem cells and induced pluripotent stem cell models have advanced rapidly and are now being used to study normal and diseased human respiratory development and lineage commitment. The differentiated progeny of iPSCs are being used as a source of normal and diseased cells to identify signaling pathways and cellular physiology of the disease as well as evaluate gene- and drug-based therapeutics for respiratory disease. Although it is likely that iPSCs may also serve as a sources of cells for replacement or regenerative therapies for lung injury or disease in the future, a significant amount of progress on the generation of specific lineages as well as extensive studies on the efficacy and safety of this approach is required before this goal is realized. Session 3: Bioengineering Approaches to Lung Regeneration: Structure and Matrix

The purpose of this session was to review recent developments in the pursuit of tissueengineered lung using decellularized lung scaffolds as the platform on which pluripotent cells can be seeded. Dame Julia Polak, M.D., D.Sc., from Imperial College London began the session by teleconferencing in to the meeting in real time from London; she reviewed the limitations of artificial membrane oxygenators and artificial lungs. She then gave an overview of the current state of the field, pointing out that acellular lung matrices used in bioreactors may be the way forward for generating bioartificial lung units. It should be noted that, after her talk, Dame Julia was able to respond to audience questions and comments over the teleconference link almost as if she was in the same room as everyone else, despite actually being separated from the rest of the audience by thousands of miles. Dr. Paolo Macchiarini from the Hospital Clinic de Barcelona then gave a presentation entitled “Cell Therapy and Bioengineered Replacement of the Airways,” in which he discussed the evidence showing that stem cells can be successfully applied to airway transplants for adults and children (37, 38). He then S33

VERMONT STEM CELL CONFERENCE extrapolated this to the vision that stem cell therapy might be extended to treat untreatable end-stage lung diseases. He pointed out that although there is still much debate about exactly which kind of cell should be used (embryonic, adult stem cells, etc.), in fact, the adult body has continual stores of stem cells in specialized niches that are regularly recruited to repair damaged tissues. Phase I-II clinical trials are now using dormant stem cells taken from a patient’s body to repair tissues and organs. This has the advantage of significantly reducing ethical and regulatory barriers to treatment. Dr. Joan E. Nichols of the University of Texas Medical Branch at Galveston presented a talk entitled “Acellular Lung Scaffolds: Influence in Cell Differentiation and Evaluations of Mechanical Integrity,” in which they focused on cell matrix interactions as critical factors in the design of functional engineered tissues. In particular, fabrication of biomaterials for use in an engineered lung must consider the role of the ECM in determining the differentiation of endothelial and epithelial cells. A major stumbling block in this field to date has been the production of suitable biocompatible scaffolding material that supports but does not restrict lung function. For example, such biomaterials must possess degradation profiles and elastic properties similar to that of normal lung ECM while avoiding the development of inflammation fibrosis. For these reasons, the way forward may be to use natural lung ECM obtained via decellularization of whole lungs (39–41). Dr. Christine Finck of the Connecticut Children’s Medical Center presented on “Embryonic Stem Cells in Tissue Engineered Scaffolds.” She began by pointing out that preterm delivery affects more than 10% of all births and accounts for 70% of perinatal mortality and that the ability to use stem cells to regenerate functional alveolar tissue could have a major impact on premature lung disease. Key to achieving this goal is to have stem cells differentiate successfully into desired cell phenotypes, something that depends on the growth factors used and the nature of the cell culture surfaces. Again, decellularizing intact lungs provides a means of obtaining culture surface having the desired properties, although the optimal approach to this has not been completely delineated. Decellularized lungs retain their S34

native collagen and elastin structures while losing nuclei and glycosaminoglycans. Inoculating decellularized rat lungs, for example, with different mixtures of fetal rat lung homogenates, endothelial cells, and A549 carcinoma cells has resulted in successful recellularization, and there is evidence that inoculation with mESC leads to differentiation into a variety of mature lung cell types. Importantly, biologic scaffolds require continuous nutrient infusion. They also reported that when sheep lungs were decellularized and orthotopically transplanted into a pneumonectomized sheep and the vasculature reanastomosed, the lungs could be ventilated without major air leaks without rupture of the scaffold. Dr. Andrew M. Hoffman of Tufts University reported on “Design of Biological Scaffolds that Promote Engraftment and Repopulation of Lung Scaffolds with Mesenchymal Stromal Cells” and began by pointing out that cultured mesenchymal stromal cells (MSCs) do not readily engraft the lung because they lack specific adherence factors for other stromal or parenchymal cells. He then focused on cell surface receptors as critical factors for the production of viable stem cell scaffolds and reported that MSCs and fibroblasts exhibit a high degree of similarity in surface phenotype, implying that universal scaffolds may be possible for MSCs. After presenting a number of research findings related to specific ECM surface receptors and associated signaling pathways, Dr. Hoffman concluded by stating that use of RGD-imbued scaffolds will likely contribute to improved engraftment in vivo and bioengineering (i.e., recellularization) of lungs ex vivo. Dr. Peter I. Lelkes of Drexel University presented on “Decellularization and Beyond: Scaffolds and Stem Cells,” focusing particularly on biomimetic scaffolds to facilitate alveolar morphogenesis and cell sourcing to populate such scaffolds. He began by discussing the use of natural and synthetic biomaterials for generating nanoscale-diameter fibers and composites for emulating the bioactive, “intelligent” structure and function of extracellular matrices. Such scaffolds can be used to manipulate various biological processes, such as the induction of branching morphogenesis. He then pointed out that cell sourcing for lung tissue engineering and regenerative pulmonary medicine may

be improved by directed differentiation and organotypic functional assembly of stem and progenitor cells. Applications include generating vascularized three-dimensional pulmonary constructs from mixed populations of fetal murine pulmonary cells, permissive natural matrices and biomaterials, and growth factor–containing culture media. Dr. Lelkes also discussed the maintenance and integration of these structures after ectopic implantation in vivo. He concluded by describing recent studies of murine embryonic stem cell differentiation in vitro. Dr. Angela Panoskaltsis-Mortari, Ph.D., from the University of Minnesota, gave a presentation entitled “Using Decellularized Matrices for Bioengineering the Lung,” in which she presented her work on acellular ECM scaffolds and their use in bioreactors to evaluate the potential of multipotent stem cells to regenerate lung tissue (42). When seeded into these scaffolds under the appropriate growth conditions, iPS can express several lung markers, which illustrates the potential for regenerating lung tissue using autologous iPS cells on decellularized matrices to avoid immune-mediated rejection on subsequent transplantation. Nevertheless, the optimization of this process will require much future research. Finally in this session, Zachary Borg, B.A., from the University of Vermont talked about “Optimizing Lung Decellularization and Recellularization with Stem Cells” and began by pointing out the current chronic shortage of donor lungs for transplant. He then summarized the current state of research into the use of decellularized lungs from autopsy seeded with stem cells to generate new lungs as well as recent results from primate and nonprimate lungs. This included a report of successful implantation of recellularized lung scaffolds into rats. Nevertheless, this field of research is still very much in its infancy, and much work remains to be done to determine the optimum conditions for regenerating lungs by recellularizing acellular scaffolds. Session 4: Bioengineering Approaches to Lung Regeneration: Function

The goal of this session was to review the progress that was made in bioengineering of the lung, with a specific focus on functional assessment of lung tissue. The

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VERMONT STEM CELL CONFERENCE session dealt with four primary topics: whole-organ engineering with decellularized scaffolds, bioinspired microdevices, mechanobiology, and advanced imaging modalities. The session began with Doris Taylor, Ph.D., who provided a general overview of the field of organ decellularization. Over the past decade, there has been a shift from the use of simple sheets of decellularized tissue to decellularization of complex tissues and organs that preserve the three-dimensional geometry of the starting tissue (39). This has been made possible by using the vasculature of the tissue or organ to deliver various decellularization agents to the cells, effectively reducing the diffusion distance and increasing the transport of cellular material from the tissue. Dr. Taylor notably pioneered this whole-organ engineering approach with the development of a decellularized rat heart (43), and a variety of other tissues have followed, including liver and lung (41, 44–46). Perfusion of the vasculature of the heart with detergents (particularly sodium dodecyl sulfate) resulted in a tissue with no visible histologic evidence of nuclei and significant DNA removal, without a significant loss of glycosaminoglycans (39). The decellularized heart served as a suitable substrate for attachment of various siteappropriate cells, including cardiomyocytes and endothelial cells. Cardiomyocytes showed isolated regions of synchronous contractility, and the addition of endothelial cells significantly reduced the thrombogenicity of the ECM scaffold. These results show that whole-organ decellularization is promising for development of a functional cardiac transplant in the future. Dr. Taylor has extended this work in the rodent model to the human case, with successful decellularization of a human heart with cardiac structure and large and small vessels left intact. There are a variety of aspects of the ECM that must be considered when selecting a donor tissue for decellularization. Ultimately, this organ decellularization and recellularization relies on the bioactivity of the matrix to allow cell attachment, proliferation, and differentiation, and these cues are dependent on the composition and passive mechanical behavior of the resulting scaffold. Studies are needed to fully describe the changes in the ECM of tissue as a function of sex, age, and disease, especially because donor tissues are likely to Vermont Stem Cell Conference

come from older individuals with more or less history of pathology. Likewise, processing has a variety of effects on the ECM that are not fully understood. Any changes to the ECM associated with these conditions could have an effect on the cells that are seeded into the construct or that migrate into the construct from the recipient. Standardization of processing techniques is going to be necessary in the near future to make reasonable comparisons. Due to the preservation of ECM structure and composition through decellularization, ECM scaffolds serve as interesting test beds for studying the potential for cells to regenerate tissue. Work by Dr. Taylor and others showed that ECM from different organs has potent effects on the gene expression of seeded cells. In particular, the gene expression of cardiomyocytes differed greatly when seeded on heart-, liver-, and kidney-derived ECM. Some combination of the specific organization of the ECM and the ECM composition appears to allow cells to retain their original phenotype. Although there is some early evidence that ECM may facilitate targeted differentiation of stem and progenitor cells, there is much work to be done to substantiate this claim (47). An important point of caution was presented regarding ECM scaffold remodeling. ECM scaffolds tend to be subject to cell infiltration followed by ECM degradation, remodeling, and deposition. This is in contrast to the desired goal of generating transplantable organs that will resume homeostasis without loss of structure. The current state of the art in this area is still too early stage to fully address this question regarding the potential for ECM scaffolds to serve as framework for future organs. Later in the session, Drs. Harald Ott, M.D., and Laura Niklason, M.D., Ph.D., both reviewed their breakthroughs in lung tissue engineering using decellularized lungs as a substrate for tissue assembly. In 2010, both groups independently engineered lung tissue and performed shortterm orthotopic implantation in rat models (48, 49). Dr. Ott focused on the many questions that will need to be addressed before widespread application of lung tissue engineering can be realized (50). His group has successfully decellularized large cadaveric lungs, but as described by Dr. Taylor, there is work yet to be done to

determine if there is an effect on the species of lung, the age of the donor, and the disease state of the tissue. Furthermore, the optimal conditions for harvesting the tissues have not been explored. It is unknown whether there are differences in the ECM from living and deceased donors or how long after death the organs persist with warm or cold ischemia before use. Furthermore, methods for organ preservation have been unexplored to date. Dr. Ott agreed that the field would need to move to a consensus about the methods for decellularization of lung tissue as well as sterilization methods. Ultimately, storage conditions and shelf life will need to be investigated. Questions regarding source and number of cells needed for repopulation of decellularized lungs loom large. Primary cells are attractive but will be difficult to obtain in sufficient numbers to generate an organ and prompt concerns about immunogenicity. Stem cells may be an alternative, but the ability to obtain enough cells remains a challenge, and there are ethical concerns about the use of embryonic and fetal stem cell populations. Induced pluripotent stem cells are another possible cells source. In all cases, methodologies will need to be developed to drive stem cells into lung cell lineages for this approach to be feasible. Furthermore, it is unclear whether stem cells should be used in their undifferentiated state and allowed to differentiate in response to the cues from the ECM and cell culture or whether they should be differentiated before seeding within the ECM. Bioreactor development is going to be another key step in the progress of organ engineering. The optimal configuration, culture protocol (e.g., media, mechanical conditioning), and monitoring regimes (e.g., metabolism, morphologic endpoints) need to be determined. The bioreactors must also be designed to be scalable not only from smaller organs to larger organs but also from single to mass production. Once all of the conditions for culture are determined, an important question will be how long the construct should be allowed to mature before implantation. The time for maturation raises the question of engineering versus regeneration. If the engineered organ is intended to experience further in situ development to gain full function, then a shorter culture period may be appropriate. This would be the S35

VERMONT STEM CELL CONFERENCE regenerative paradigm. In contrast, tissue engineering would reach for the goal of a transplantable organ that would resume homeostasis on implantation. For this approach, a much longer and more involved culture system would be required. Finally, determining the most appropriate model for transplantation will be crucial to have the best chance of success. Animal models will be necessary to enable determination of the effects of the immune response to the graft and whether homeostasis can be achieved. It will also be necessary to determine whether the grafts can tolerate ventilatory support. Depending on how quickly mucociliary clearance function is restored, provisions may need to be made for palliative care. Dr. Ott’s most recent work drives home the need for further investigation of these questions (50). Building on his initial work, the model was expanded to include evaluation of orthotopic implanted single lobe grafts over a period 14 days. The engineered lung constructs showed similar oxygen transport capacity as comparable to a cadaveric lung transplant for a period of 7 days, but function declined over the second week due to graft consolidation and inflammation. By the end of the study, the engineered lung construct showed no ventilation or clearance and showed evidence of infection. Dr. Niklason shared her progress in the development of functional lung constructs. Dr. Niklason described the use of decellularization methods based primarily on the use of vascular and airway perfusion with CHAPS detergent that has been shown to preserve the structure of the lung ECM (48, 51). The cell source used by Dr. Niklason was a mixed population of neonatal rat lung cells. Based on histologic data defining the phenotypes of the cells and the composition of the adjacent matrix, it was found that either the mixed cell populations tended to sort themselves to “zip codes” in the matrix, or the cells attached randomly to the ECM and remodeled it to generate an appropriate niche. The cells tended to show increased expression of regenerative markers in the early time frame after seeding into ECM scaffolds, and over a period of 12 days the phenotype changed to express more terminally differentiated, site-appropriate cells. This was all performed in Dulbecco’s modified Eagle medium with 10% fetal bovine serum and no other airway-specific S36

markers, suggesting that the ECM is able to drive tissue assembly. The next major thrust of this section dealt with bioinspired microdevices. The first talk was by Dr. David Hoganson, who described his work in the development of microfluidic systems to generate a lung assist device (52). Lung assist devices have been in development for decades, and the first device (Novalung) is now being used in the United States (53). The typical design for these devices use high-density parallel hollow fibers, which poses significant deviations from the physiologic flow in bifurcated vascular networks of the lungs. Using microfluidics, very small channels were formed by polydimethylsiloxane onto silicon molds by lithography to establish a vascular network and alveoli chamber separated by a gaspermeable membrane. The design of the vascular network allowed for dramatic increase is surface area, which is critical for adequate gas exchange, while maintaining physiologic flow and shear conditions to prevent thrombosis. The three-dimensional vascular networks can be micromachined with approximately 2-mm precision. Empirical experiments have shown that the results match the predictions for computation fluid dynamics analysis. Further work is being performed to optimize the gas transport kinetics by modifying the channel shape and size, and selection of different substrates (54–56). For example, the use of PTFE films was found to increase the carbon dioxide permeability by 300 times. The empirical studies also showed the importance of manufacturing, as a small manufacturing defect led to an increased platelet adhesion and thrombosis. Future developments will also include biomimetic scaffolds to enhance endothelialization of the channels. Dr. Daniel Huh presented his work on the development of a “breathing lung on a chip” (57, 58). This approach also used photolithography to form microfluidic channels from silicone but takes advantage of the natural elasticity of silicone to allow for application of stretch to cells within the system. This allows for testing lung cells in a system that mimics the stretch of respiration. The configuration of the system included alveolar epithelial cells on the air side of the membrane and microvascular endothelial cells on the fluid side of the membrane. The membrane had micropores to allow for transport

across the membrane. Unlike the approach described by Hoganson and colleagues, which was primarily intended to support oxygenation function for a diseased lung, this “lung on a chip” approach was intended to recapitulate the organ level function of the lung in a very small scale. Studies showed that the configuration was able to restore alveolar—capillary barrier function and that the morphology of the cells in response to mechanical loading mimicked that observed in vivo. Furthermore, activation of endothelial cells with tumor necrosis factor-a led to adhesion of circulating neutrophils within the microchannels. Neutrophils were then shown to transmigrate to the apical surface of the system. The system was further extended to include bacteria within the apical compartment that induced activation of the endothelium followed by attachment and transmigration of neutrophils. The system was also tested with various nanoparticles to test toxicity. Silica nanoparticles and carboxylated Cd/Se quantum dots were both shown to induce inflammation and increases in reactive oxygen species production but only in the presence of accompanying mechanical strain. The mechanical strain–induced response appeared to be specific to these nanoparticles, as other nanomaterials did not show the same effect. This suggested that care must be taken when deciding whether to use mechanical ventilation in patients exposed to these materials and demonstrated the potential for these microdevices to impact clinical care. Dr. Dan Tschumperlin, Ph.D., followed with a broader look at recent developments in mechanobiology of the lung, in particular the importance of matrix stiffness and the response of cells to stiffness (59). Recent advances in imaging techniques have made it possible to interrogate the micromechanics of the lung as never before. A recent report using optical sectioning microscopy showed the heterogeneous distention of alveolar segments in response to inflation, with significantly greater distention of type 1 epithelial cells as compared with type 2 cells (60). This finding emphasized the importance of the composition, structure, and stiffness of the ECM on the phenotype and behavior of cells. Dr. Tschumperlin’s group recently measured the stiffness of normal and fibrotic mouse lung matrix on the microlevel using atomic force

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VERMONT STEM CELL CONFERENCE microscopy and investigated the effects of stiffness on lung fibroblasts (61). ECM in fibrotic lesions showed a sixfold higher stiffness than in the surrounding lung parenchyma, which is a larger difference than observed in macroscale measurements. In in vitro experiments, the stiffness levels corresponding to normal lung parenchyma (z0.5 kPa) led to more rounded cells that tended to show decreased proliferation, increased apoptosis, and decreased collagen synthesis. In contrast, stiffness levels ranging from 3 to 15 kPa, which corresponded to the stiffness of fibrotic lesions, showed the opposite trends. Increased stiffness also led to decreased matrix metalloproteinase expression. It is important to note that the shift toward more fibrotic fibroblast phenotype occurred at stiffness levels just slightly above those observed in normal tissue, suggesting that the native ECM suppresses fibrosis, and only a slight injury could perpetuate the formation of the lesion. PGE2 was found to mediate the fibroblast response to stiffness. ROCK down-regulated COX-2 to suppress PGE2 synthesis, thereby leading to a more fibrotic fibroblast phenotype. However, not all cells respond to stiffness in the same way. Another study investigated the response of various cancer cells and fibroblasts to varying stiffness, and “rigidity-dependent” and “rigidity-independent” growth profiles were observed, with low growth rates on soft gels due in part to selective alterations in the cell cycle progression (62). The rigiditydependent cells also tended to show less spreading and migration on soft gels. On implantation, the rigidity-independent lines were able to grow more robustly on normal lung matrix. Clearly, matrix stiffness and mechanobiology are important areas for continued investigation in lung biology. Finally, Dr. Jason Woods, Ph.D., presented developments in in vivo imaging of the lung. Assessment of conditions like inflammation can only be tested indirectly through pulmonary function testing or through invasive bronchoalveolar lavage. The current state of the art in evaluating lung morphology is histologic analysis, which has the limitations of being constrained to two-dimensional planar sections of tissues and is destructive in nature. In both cases, new minimally invasive imaging modalities that allow for three-dimensional visualization of the lung would be advantageous. A recent study Vermont Stem Cell Conference

evaluated the potential for positron emission tomographic imaging with [18F] fluorodeoxyglucose ([18F]FDG) to monitor inflammation in the lung (63). A small volume of endotoxin was instilled into a limited region of the lungs of healthy patients. At 24 hours after endotoxin instillation, [18F]FDG–positron emission tomographic was performed and was able to show with acuity the presence of inflammation at the instillation site. The rapid uptake of [18F]FDG for acute and chronic inflammation suggests that it will be a useful biomarker to assess the efficacy of antiinflammatory therapies in conditions like CF, ARDS, and pneumonia. Another technique described by Dr. Woods for visualizing the lung microstructure was hyperpolarized gas magnetic resonance imaging. Hyperpolarized magnetic resonance imaging with 3He or 129Xe works by measuring the random atomic motion (diffusion) of the gas molecules (64, 65). Because airway structures will limit the diffusion of gas, measurement of the atomic motion will provide information about the lung microstructure. Both gases have been used in humans, large animals, and, more recently, small animals in both healthy and diseased conditions and have provided important insights into the three-dimensional structure of the lungs (64–73). The usefulness of this approach to treatment of human disease is obvious, with greater ability to determine the changes in lung microstructure as a function of disease and over time. The expansion of this technique to mouse models opens up huge opportunities to study the architectural changes in the lung as a function of disease and treatment with all of the advantages of small animal models. This session showed how advances in the field of regenerative medicine, microscopy, and imaging are changing paradigms regarding the study the mechanisms and progression of disease and development of strategies to repair or replace injured lung tissue. These new approaches are providing opportunities for the field to move toward a more rigorous study of the structure–function relationships in the lung. Session 5: EPCs, MSCs, and Cell Therapy Approaches for Lung Diseases

The general theme for this session was the evolving use of MSCs and EPCs for the prevention and/or treatment of important

diseases of the lung, ranging from ALI/ ARDS to pulmonary hypertension. This session primarily focused on the potential of these “stem” cell populations to function in immunomodulatory and/or tissue repair roles rather than on their regenerative medicine capacities per se. The first speaker in the session was Dr. Darwin Prockop, M.D., Ph.D. (Texas A&M Health Science Center College of Medicine, Temple, TX) who addressed the question of whether studies using therapeutic administration of MSCs could ultimately translate to the development of novel protein or small molecule therapies for ALI. Dr. Prockop began his presentation with the observation that although MSCs have been reported to produce beneficial effects in a wide variety of disease models, the precise mechanism(s) by which MSCs exert these effects remain(s) unclear. He suggested three possible paradigms that could be responsible for the observed beneficial effects: (1) MSCs could provide a microenvironment or “niche” providing cell-to-cell contact to nourish resident cells, (2) MSCs could engraft and differentiate to replace injured cells in the target tissue (i.e., regenerative medicine), or (3) MSCs could express and secrete/ release specific gene products with the potential to decrease or reverse diseaserelated pathophysiology in the host via a paracrine mechanism. Dr. Prockop considered paradigm one to be highly unlikely to account for MSC beneficial effects in lung diseases. Although intravenously administered MSCs are initially “trapped” in the lung, imaging studies have failed to document significant long-term pulmonary engraftment of MSCs (74, 75). In the lung, he believes that paradigm three is most likely, and the identification of factors secreted by MSCs may enable the development of novel specific protein- or small molecule–based therapies for ALI. His group has recently identified two new MSC-derived gene products as candidate molecules. TSG-6 is an antiinflammatory protein secreted by MSCs that has been shown to play an important role in MSC-mediated improvement of experimental myocardial infarction in the mouse (76). A subsequent study demonstrated that MSC-derived TSG-6 attenuates experimental zymosaninduced peritonitis via a paracrine effect to decrease proinflammatory responses in S37

VERMONT STEM CELL CONFERENCE resident macrophages activated by Toll-like receptor 2/nuclear factor-kB signaling (77). The second recently identified MSCderived molecule discussed by Dr. Prockop was stanniocalcin 1 (STC-1), which has been shown to have antioxidant and antiapoptotic properties (78). Dr. Prockop concluded with his opinion that a careful and comprehensive analysis of the activated MSC transcriptome and “secretome” (i.e., secreted gene products) is likely to yield potential candidates for translational development of novel protein-based strategies for the treatment of ALI. It is highly possible that the beneficial effects of MSCs in diseases associated with inflammation and tissue injury are not due to a single soluble factor secreted by MSCs but rather to a combination of MSCderived factors that target various components in the pathophysiology of the disease process. Furthermore, MSCs may have to be activated by the host microenvironment to express specific factors responsible for their beneficial immunomodulatory and/or reparative properties. Dr. Prockop also mentioned that he now works exclusively with human MSCs in mouse models, due to the inherent instability of mouse-derived MSCs and associated inconsistent experimental results. Dr. Michael Matthay, another speaker in this section (see below), commented that his group also uses human MSCs in mouse models of ALI (and other mammalian models of ALI) with consistent therapeutic effects. The next speaker in this session was Dr. Jacques Galipeau (Emory University Winship Cancer Institute, Atlanta, GA), who provided a cautionary discussion of the immune plasticity of MSCs and the associated impact on the design of MSCbased preclinical and clinical studies. He emphasized that human and rodent MSCs differ in their respective immune properties and urged caution when attempting to translate findings in murine models to clinical settings, such as graft-versus-host disease and Crohn disease (79, 80). Furthermore, although MSCs are generally viewed as nonimmunogenic, “immunoprivileged” cells with immunosuppressive and antiinflammatory properties suitable for use in allogeneic cell therapy, there is evidence that MSCs can function under certain circumstances as antigen-presenting cells and effectively S38

promote alloimmunization-type responses and even lead to loss of tolerance to selfantigen (79, 81–87). Because these observations have serious implications for the use of MSCs in clinical cell-based therapeutic regimens, Dr. Galipeau suggested that further consideration be given to strategies using autologous human MSCs in clinical trials. However, loss of antiinflammatory and/or immunosuppressive functional activity in stored (e.g., cryopreserved) human MSCs represents a potential hurdle of major significance in the development of autologous MSC therapy. Data were presented demonstrating that it may take 24 hours or longer after thawing for cells to regain full function. Dr. Donald Phinney (Scripps Research Institute, Jupiter, FL) expanded on the implications of functional heterogeneity in MSCs for clinical therapies in his presentation. He and his colleagues have developed high throughput assays to evaluate and quantify the trilineage differentiation potential of human MSCs at the clonal level (88). These assays demonstrated that human MSCs represent a heterogeneous population of clonal progenitors with varying potency and biological/physiological properties. Functional studies revealed clonal variation in growth and survival rates, which correlated with differences in the expression of TWIST, TWIST2, and BMI1 (88, 89). In a severe combined immunodeficiency mouse model of ALI, Phinney and colleagues have observed that secretion of IL-1 receptor antagonist (IL-1RA) plays an important role in the ability of MSCs to improve outcome (89). This antiinflammatory activity was suppressed by pretreatment of MSCs with FGF2, which suppressed expression of TWIST. These findings suggest a critical role for TWIST in the regulation of multipotent clonal expansion and antiinflammatory activity in MSCs. Dr. Phinney emphasized that variable biologic capacities in MSCs can have serious consequences for studies of their potential for the treatment of disease states. The biological properties and behavior of MSCs are influenced by media supplements used in the cultivation of MSCs, level of expansion of MSCs, and the initial plating density of MSCs. As of July 2011, more than 180 trials using MSCs have been completed or are underway. Variable, and sometimes inconsistent, degrees of

therapeutic efficacy have been reported. The selection of specific clones during isolation, expansion, and cultivation in vitro can critically affect subsequent functions of MSCs in both preclinical studies and clinical trials. Dr. Michael Matthay (University of California—San Francisco, San Francisco, CA) discussed studies performed by his group investigating the potential therapeutic use of allogeneic human MSCs (hMSCs) for the treatment of ALI. In a murine model of experimental gramnegative pneumonia, hMSCs administered either intravenously or intratracheally reduced the severity of ALI (including modulation of proinflammatory responses and restoration of normal lung fluid balance), and improved survival (90). MSCs also enhanced the phagocytic activity of resident monocytes/macrophages, resulting in enhanced bacterial clearance in vivo consistent with findings recently reported in experimental sepsis (91, 92). Another mechanism contributing to hMSCmediated enhancement of bacterial clearance is the release of soluble antimicrobial peptides, including LL-37 (cathelicin B and lipocalin-2) by MSCs (93, 94). In a study of ex vivo perfused lung, intravenous hMSCs reduced lung endothelial permeability changes induced by either LPS or live Escherichia coli, primarily via antiinflammatory and tissue reparative paracrine mechanisms involving the release of hMSC-derived factors, including keratinocyte growth factor, angiopoietin-1, and the aforementioned antimicrobial peptides (93, 95). Thus, multiple pathways are likely responsible for the observed hMSC-mediated treatment effects in ALI and sepsis, including pathways that reduce inflammation and tissue injury as well as pathways that enhance repair of damaged endothelium and lung epithelium. In collaboration with Dr. Dan Traber (University of Texas—Galveston, Galveston, TX), Dr. Matthay and colleagues are currently completing evaluation of hMSCs in a large animal model of ALI in sheep. These studies involve measurement of pulmonary and systemic hemodynamics and respiratory function in preparation for a clinical trial testing clinical grade cryopreserved allogeneic hMSCs (supplied by the NHLBI-sponsored Production Assistance for Cell Therapies [PACT] Program at the University of Minnesota)

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VERMONT STEM CELL CONFERENCE for the treatment of severe ALI/ARDS. This planned clinical trial would focus on the safety and respiratory efficacy endpoints, such as oxygenation and pulmonary dead space fraction. Next, Dr. Sam Janes (University College London, London, UK) presented work from his laboratory investigating the use of MSCs to deliver gene therapies for the treatment of cancer involving the lung. Dr. Janes presented data showing that lentiviral vectors can be used to transduce MSCs effectively and safely. Using this system, he discussed studies using MSCs engineered to express the proapoptotic molecule TRAIL in an inducible manner using the TET-ON regulation system to kill cancer cells both in vitro and in vivo in a mouse model of metastatic lung disease (96–98). Further discussion focused on the regulatory hurdles and advancements needed to translate this novel type of antitumor therapy in the clinical setting (96). Dr. Meagan Goodwin and Dr. Daniel Weiss (University of Vermont, Burlington, VT) addressed the use of MSCs for the treatment of obstructive airway diseases in the next portion of this session. Specifically, the reported beneficial immunomodulatory effects of MSC therapy were discussed in the context of rodent models of asthma, bronchiolitis obliterans (BO), and COPD (99–122). A small clinical trial (10 patients) investigating the potential of MSCs, collected from related or unrelated HLA-identical or HLAmismatched donors, to treat BO after lung transplantation is ongoing at Prince Charles Hospital, Perth, Australia. A clinical trial of autologous MSCs in COPD has been initiated at the University of Leiden (The Netherlands) examining the effect of administration of autologous MSCs before lung volume reduction surgery for severe pulmonary emphysema. Finally, Dr. Weiss served as an investigator on a phase II, multicenter, double-blind, placebo-controlled, industry-sponsored (Osiris Therapeutics, Inc.) clinical trial of allogeneic MSCs to treat moderate to severe COPD. This recently completed study randomized 62 patients from six sites to MSC therapy versus placebo. The patients received a total of four intravenous infusions over the course of 4 months and were followed for 2 years after the first infusion (4). The data are currently being analyzed, but Dr. Weiss reported that no Vermont Stem Cell Conference

safety concerns arose during the course of the trial. He concluded that obstructive airway diseases are viable targets for MSCbased therapeutics. In addition, he emphasized that mechanistic studies with proper controls are essential in both preclinical and clinical studies to further advance the field. The next speakers in this session were Dr. Mervin Yoder (Indiana University School of Medicine, Indianapolis, IN) and Dr. Bernard Thebaud (University of Alberta, Edmonton, AB, Canada), who spoke on the topic of human EPCs. Dr. Yoder focused on the controversies surrounding the definition, biological properties, and functions of EPCs (123– 125). The term EPC was first used more than 15 years ago to refer to circulating cells with endothelial cell–like characteristics and surface markers in the human bloodstream that could augment blood flow to sites of experimental ischemia in rodents. Subsequent studies have defined a number of subtypes of circulating EPCs in normal human subjects and in individuals with a variety of clinical disorders. Most investigators attribute the hematopoietic component of the bone marrow as the origin of EPCs. It appears as though the vast majority of EPCs represent cells of the hematopoietic lineage, but most current evidence indicates that EPCs do not differentiate into functional endothelial cells. Rather than serving as endothelial cell progenitors per se, EPCs exhibit proangiogenic activity and function to promote vascular regeneration. Dr. Yoder suggested that a number of alternative designations better define the vast majority of cells classified in the literature as EPCs. More accurate, alternative terms for EPCs include circulating progenitor cells, circulating angiogenic cells, circulating proangiogenic hematopoietic stem and progenitor cells, circulating myeloid cells, and Tie2-expressing macrophages. Dr. Yoder presented new evidence that a very rare circulating endothelial colony forming cell (ECFC) does exist with clonal proliferative potential, cell surface antigens characteristic of mature endothelial cells but not blood cells, and capacity to form capillary lumens in vitro and blood vessel formation in vivo. He concluded his talk with recent data demonstrating that ECFCs form human vessels in vivo when injected into immunodeficient mice. The physiological properties of the ECFC-

derived capillary plexus were shown to be modulated by ECM, mural cells, and Notch signaling. Dr. Thebaud discussed the potential use of ECFCs to treat bronchopulmonary dysplasia (BPD) in newborns (126–134). He presented evidence that ECFCs exist in the developing lung and that ECFC function is impaired in experimental O2-induced BPD. Furthermore, human umbilical cord–derived ECFCs were sown to lung structure and function and decrease pulmonary hypertension in experimental O2-induced BPD. He concluded that ECFCs represent a promising novel treatment strategy for clinical BPD and related disorders characterized by arrested alveolar development and pulmonary hypertension. This session concluded with a talk presented by Dr. Duncan Stewart (Ottawa Hospital Research Institute—University of Ottawa, Ottawa, ON, Canada), who presented an update on EPC-based therapies for pulmonary arterial hypertension (PAH) (135). In preclinical studies, administration of syngeneic EPCs can prevent the development of experimental PAH in the rat. More importantly, EPCs transfected with endothelial NO-synthase (eNOS) were able to reverse established PAH and prolong survival (136). Dr. Stewart is currently directing a “first-in-human” dose escalation study, the Pulmonary Hypertension and Cell Therapy (PHACeT) trial, investigating the safety of eNOS-transfected autologous EPCs in patients with severe, refractory PAH. Although enrollment in this singlecenter trial has proven challenging, preliminary data suggest potential improvements in functional and hemodynamic parameters in treated patients. These improvements appear to be related to NO, the eNOS transgene product. These promising results have spurred planning for an upcoming, rigorously designed randomized, blinded phase II clinical trial in patients with PAH to be performed in multiple centers across North America (PHACeT-2). Session 6: Perspectives from the NHLBI, FDA, and Respiratory Disease Foundations: Summation and Directions

Dr. Carol Blaisdell, National Heart, Lung and Blood Institute. “Building the Foundation for Lung Regeneration— S39

VERMONT STEM CELL CONFERENCE NHLBI Perspective” described the NHLBIsupported science in lung progenitor/stem cell biology, which consisted of less than a handful of projects in 2001 and has grown to nearly 70 grants in fiscal year 2010. The program is examining several aspects of lung stem cells, such as the pluripotency of lung resident cells during development and after lung injury, the impact of mesenchymal stromal cells in preclinical studies of lung repair, and the potential of decellularized scaffolds for lung regeneration. Clinical research in lung stem cell therapies along with the knowledge gained by understanding the basic biology of the lung stem cell populations will be needed to translate to diagnostic and therapeutic strategies in the future. Dr. Amy I. Farber, Ph.D., LAM Treatment Alliance. “Return on Investment: A Case Study in Driving Science Focused on Patient Impact” described that lymphangioleiomyomatosis (LAM) is a multisystem disease predominantly affecting women of childbearing age. LAM occurs as a sporadic disorder or in association with tuberous sclerosis complex (TSC), a genetic disease affecting multiple organs including the lungs, kidneys, skin, eyes, and brain. TSC and LAM have been associated in some cases with mutations in the TSC1 or TSC2 tumor suppressor genes. Pulmonary LAM is likely more sex-specific than breast cancer. LAM is frequently fatal due to respiratory failure caused by worsening airflow obstruction. The LAM Treatment Alliance is the largest LAM research organization in the world, driving an aggressively funded, highimpact research strategy and fostering innovative patient partnerships to effectively treat and eradicate LAM in time for patients living with the disease today. The Alliance is hopeful that collaborations with those working in the stem cell area will provide insights into key discovery and engineering efforts focused on overcoming hurdles and answering questions regarding the cell of origin, challenges to isolating, growing, and analyzing LAM cells, and tissue- and cell-based therapeutics. Dr. Daniel M. Rose, M.D., CEO, Pulmonary Fibrosis Foundation (PFF), described that the PFF was founded in September 2000, with missions of finding a cure for IPF, advocating for the pulmonary S40

fibrosis community, promoting disease awareness, and providing a compassionate environment for patients and their families. IPF is a chronic diffuse interstitial lung disease of unknown etiology characterized by inflammation and fibrosis of the lung parenchyma. The disease is probably underdiagnosed and underreported. In the United States, the prevalence has been estimated at 56 cases per 100,000 individuals, with a slightly higher incidence among men. The mortality rate at 5 years is estimated to be 50 to 70%. The diagnosis can be made with high-resolution computed tomography scanning or surgical lung biopsy. Biopsy specimens from individuals with IPF typically will show the histological appearance of usual interstitial pneumonia. It is hypothesized that the disease is associated with active alveolar injury and abnormal wound healing. Presently there are no effective pharmacological therapies. Dr. Donald W. Fink, Jr., Ph.D., Center of Biologics Evaluation and Research, FDA. “FDA Perspective: Stem Cell–Based Products and Lung Disease” described the expectation that cellular biologic therapies consisting of or derived from ESCs, fetal stem cells, adult stem cells, or iPSCs (stem cell–based products) will provide effective treatments for a variety of current unmet medical conditions including debilitating lung diseases continues to drive expanding research efforts. Evaluating the safety profile of an investigational stem cell– based product intended for early-phase clinical testing is the responsibility of the FDA Office of Cellular, Tissue, and Gene Therapies, which possesses more than 2 decades’ experience regulating cellular therapies, including stem cell–based products. Since conclusion of the 2009 conference, the FDA has received and reviewed numerous investigational new drug applications involving stem cell– based therapies for a number of indications, including critical limb ischemia, ischemic stroke, spinal muscular atrophy, articular cartilage defects, macular degeneration, spinal cord injury, healing of chronic wounds, and cardiac disease. Notably absent are early-phase clinical safety trials for lung disease. Recent publication of evidence for the presence of resident human lung stem cells could spur progress in this area of unmet medical need.

Dr. Adam Wanner, M.D., Alpha-1 Foundation. “Role of Venture Philanthropy in Finding New Therapeutic Solutions for Alpha-1 Antitrypsin Deficiency” described that a1-antitrypsin deficiency (Alpha-1) is a rare genetic disorder that can lead to potentially fatal chronic lung disease in adults and chronic liver disease in children and adults. Although currently available therapies may halt the progression of lung disease, there are no effective treatments for the liver disease, and a cure for Alpha-1 remains elusive. Academic research, to a great extent supported by the Alpha-1 Foundation, has identified novel therapeutic targets but is not in the business of developing new therapies. Big pharmaceutical companies, on the other hand, have the means of introducing new drugs and other treatments to the market but until now have been reluctant to invest in new therapeutic solutions for rare diseases. The Alpha-1 Project, the Alpha-1 Foundation’s venture philanthropy program, is designed to accelerate the process of new drug development and ultimately develop a cure for Alpha-1. The rationale for the program is based on exciting recent research that demonstrates how the lung and liver disease of Alpha-1 come about and on the biotechnology community’s growing interest in Alpha-1 and its treatment. The Alpha-1 Project website (www.thealpha1project.com) provides additional information on the program and how to apply for funding.

Summary and Conclusions A continuing accumulation of data in both animal models and clinical trials suggests that cell-based therapies and novel bioengineering approaches may be potential therapeutic strategies for lung repair and remodeling after injury. In parallel, further understanding of the role of endogenous lung progenitor cells will provide further insight into mechanisms of lung development and repair after injury and may also provide novel therapeutic strategies. Remarkable progress has been made in these areas since the last conference 2 years ago. It is hoped that the workshop recommendations (Table 2) will spark new research that will provide further understanding of mechanisms of repair of

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VERMONT STEM CELL CONFERENCE lung injury and further provide a sound scientific basis for therapeutic use of stem and cell therapies in lung diseases. n Author disclosures are available with the text of this article at www.atsjournals.org. The Chairmen Gratefully Acknowledge the Program Design Assistance Received: Bioengineering Joaquin Cortiella, M.D., M.P.H. Christine Finck, M.D. Thomas Gilbert, Ph.D. David Hoganson, M.D. Peter Lelkes, Ph.D. Joan Nichols, Ph.D. Laura Niklason, M.D. Angela Panoskaltsis-Mortari Ph.D. Julia Polak, D.B.E., M.D., D.Sc.

MSCs/ESCs/Clinical Trials Armand Keating, M.D. W. Conrad Liles, M.D., Ph.D. Michael Matthay, M.D. Duncan Stewart, M.D. Mervin Yoder, M.D. Endogenous Progenitors/ESCs/iPS Cells Ivan Bertoncello, Ph.D. Carolyn Lutzko, Ph.D. Darrell Kotton, M.D. Carla Kim, Ph.D. Jay Rajagopal, M.D. Emma Rawlins, Ph.D. Sue Reynolds, Ph.D. Barry Stripp, Ph.D. Rick Wetsel, Ph.D. A list of participants, travel award winners, executive summaries of speaker

References 1 Weiss DJ, Berberich MA, Borok Z, Gail DB, Kolls JK, Penland C, Prockop DJ; NHLBI/Cystic Fibrosis Foundation Workshop. Adult stem cells, lung biology, and lung disease. Proc Am Thorac Soc 2006;3:193–207. 2 Weiss DJ, Kolls JK, Ortiz LA, Panoskaltis-Mortari A, Prockop DJ. Stem cells and cell therapy approaches for lung diseases. Conference report. Proc Am Thorac Soc 2008;5:637–667. 3 Weiss DJ, Bertoncello I, Borok Z, Kim C, Panoskaltsis-Mortari A, Reynolds S, Rojas M, Stripp B, Warburton D, Prockop DJ. Stem cells and cell therapies in lung biology and lung diseases. Proc Am Thorac Soc 2011;8:223–272. 4 Weiss DJ, Casaburi R, Flannery R, LeRoux-Williams M, Tashkin DP. A placebo-controlled randomized trial of mesenchymal stem cells in chronic obstructive pulmonary disease. Chest 2013;143:1590–1598. 5 Winkelmann A, Noack T. The Clara cell: a “Third Reich eponym”? Eur Respir J 2010;36:722–727. 6 American Thoracic Society. 2013 Nomenclature Changes [accessed May 1, 20130]. Available from: http://www.atsjournals.org/page/ nomenclature_2013 7 Rawlins EL, Hogan BL. Epithelial stem cells of the lung: privileged few or opportunities for many? Development 2006;133:2455–2465. 8 Giangreco A, Arwert EN, Rosewell IR, Snyder J, Watt FM, Stripp BR. Stem cells are dispensable for lung homeostasis but restore airways after injury. Proc Natl Acad Sci USA 2009;106:9286–9291. 9 Evans MJ, Cabral-Anderson LJ, Freeman G. Role of the Clara cell in renewal of the bronchiolar epithelium. Lab Invest 1978;38:648–653. 10 Reynolds SD, Giangreco A, Power JH, Stripp BR. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration. Am J Pathol 2000;156: 269–278. 11 Hegab AE, Ha VL, Gilbert JL, Zhang KX, Malkoski SP, Chon AT, Darmawan DO, Bisht B, Ooi AT, Pellegrini M, et al. Novel stem/ progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential. Stem Cells 2011;29: 1283–1293. 12 Ghosh M, Brechbuhl HM, Smith RW, Li B, Hicks DA, Titchner T, Runkle CM, Reynolds SD. Context-dependent differentiation of multipotential keratin 14-expressing tracheal basal cells. Am J Respir Cell Mol Biol 2011;45:403–410. 13 Kajstura J, Rota M, Hall SR, Hosoda T, D’Amario D, Sanada F, Zheng H, Ogorek ´ B, Rondon-Clavo C, Ferreira-Martins J, et al. Evidence for human lung stem cells. N Engl J Med 2011;364:1795–1806. 14 Tsao PN, Wei SC, Wu MF, Huang MT, Lin HY, Lee MC, Lin KM, Wang IJ, Kaartinen V, Yang LT, et al. Notch signaling prevents mucous metaplasia in mouse conducting airways during postnatal development. Development 2011;138:3533–3543.

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presentations, and poster abstracts are included in the accompanying online supplement. Acknowledgment: The organizers thank the Alpha-1 Foundation, American Thoracic Society, LAM Treatment Alliance, Pulmonary Fibrosis Foundation, University of Vermont College of Medicine, University of Vermont Department of Medicine, and the Vermont Lung Center for financial support for the conference. They also thank the staffs of the University of Vermont Continuing Medical Education and University of Vermont College of Medicine Communications Offices, notably Terry Caron, Natalie Remillard, Jennifer Nachbur, and Carole Whitaker, for organizational support and Gwen Landis for administrative support.

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VERMONT STEM CELL CONFERENCE 28 Gjorevski N, Nelson CM. Bidirectional extracellular matrix signaling during tissue morphogenesis. Cytokine Growth Factor Rev 2009; 20:459–465. 29 McQualter JL, Bertoncello I. Concise review: deconstructing the lung to reveal its regenerative potential. Stem Cells 2012;30:811–816. 30 Brechbuhl HM, Ghosh M, Smith MK, Smith RW, Li B, Hicks DA, Cole BB, Reynolds PR, Reynolds SD. b-catenin dosage is a critical determinant of tracheal basal cell fate determination. Am J Pathol 2011;179:367–379. 31 Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, Crowley D, Bronson RT, Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005;121:823–835. 32 Somers A, Jean JC, Sommer CA, Omari A, Ford CC, Mills JA, Ying L, Sommer AG, Jean JM, Smith BW, et al. Generation of transgenefree lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 2010;28:1728–1740. 33 Samadikuchaksaraei A, Bishop AE. Derivation and characterization of alveolar epithelial cells from murine embryonic stem cells in vitro. Methods Mol Biol 2006;330:233–248. 34 Samadikuchaksaraei A, Cohen S, Isaac K, Rippon HJ, Polak JM, Bielby RC, Bishop AE. Derivation of distal airway epithelium from human embryonic stem cells. Tissue Eng 2006;12:867–875. 35 Longmire TA, Ikonomou L, Hawkins F, Christodoulou C, Cao Y, Jean JC, Kwok LW, Mou H, Rajagopal J, Shen SS, et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 2012;10:398–411. 36 Wong AP, Bear CE, Chin S, Pasceri P, Thompson TO, Huan LJ, Ratjen F, Ellis J, Rossant J. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat Biotechnol 2012;30:876–882. 37 Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, Dodson A, Martorell J, Bellini S, Parnigotto PP, et al. Clinical transplantation of a tissue-engineered airway. Lancet 2008;372: 2023–2030. 38 Jungebluth P, Alici E, Baiguera S, Le Blanc K, Blomberg P, Bozoky ´ B, Crowley C, Einarsson O, Grinnemo KH, Gudbjartsson T, et al. Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study. Lancet 2011;378:1997–2004. 39 Badylak SF, Weiss DJ, Caplan A, Macchiarini P. Engineered whole organs and complex tissues. Lancet 2012;379:943–952. 40 Panoskaltsis-Mortari A, Weiss DJ. Breathing new life into lung transplantation therapy. Mol Ther 2010;18:1581–1583. 41 Nichols JE, Niles JA, Cortiella J. Production and utilization of acellular lung scaffolds in tissue engineering. J Cell Biochem 2012;113: 2185–2192. 42 Price AP, England KA, Matson AM, Blazar BR, Panoskaltsis-Mortari A. Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded. Tissue Eng Part A 2010;16:2581–2591. 43 Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, Taylor DA. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 2008;14:213–221. 44 Krawiec JT, Vorp DA. Adult stem cell-based tissue engineered blood vessels: a review. Biomaterials 2012;33:3388–3400. 45 Totonelli G, Maghsoudlou P, Garriboli M, Riegler J, Orlando G, Burns AJ, Sebire NJ, Smith VV, Fishman JM, Ghionzoli M, et al. A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration. Biomaterials 2012;33: 3401–3410. 46 Fishman JM, De Coppi P, Elliott MJ, Atala A, Birchall MA, Macchiarini P. Airway tissue engineering. Expert Opin Biol Ther 2011;11: 1623–1635. 47 Cortiella J, Niles J, Cantu A, Brettler A, Pham A, Vargas G, Winston S, Wang J, Walls S, Nichols JE. Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation. Tissue Eng Part A 2010;16:2565–2580. 48 Petersen TH, Calle EA, Zhao L, Lee EJ, Gui L, Raredon MB, Gavrilov K, Yi T, Zhuang ZW, Breuer C, et al. Tissue-engineered lungs for in vivo implantation. Science 2010;329:538–541.

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49 Ott HC, Clippinger B, Conrad C, Schuetz C, Pomerantseva I, Ikonomou L, Kotton D, Vacanti JP. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med 2010;16:927–933. 50 Song JJ, Kim SS, Liu Z, Madsen JC, Mathisen DJ, Vacanti JP, Ott HC. Enhanced in vivo function of bioartificial lungs in rats. Ann Thorac Surg 2011;92:998–1005, discussion 1005–1006. 51 Petersen TH, Calle EA, Colehour MB, Niklason LE. Matrix composition and mechanics of decellularized lung scaffolds. Cells Tissues Organs 2012;195:222–231. 52 Hoganson DM, Pryor HI II, Vacanti JP. Tissue engineering and organ structure: a vascularized approach to liver and lung. Pediatr Res 2008;63:520–526. 53 Gazit AZ, Sweet SC, Grady RM, Huddleston CB. First experience with a paracorporeal artificial lung in a small child with pulmonary hypertension. J Thorac Cardiovasc Surg 2011;141:e48–e50. 54 Kung MC, Lee JK, Kung HH, Mockros LF. Microchannel technologies for artificial lungs: (2) screen-filled wide rectangular channels. ASAIO J 2008;54:383–389. 55 Lee JK, Kung HH, Mockros LF. Microchannel technologies for artificial lungs: (1) theory. ASAIO J 2008;54:372–382. 56 Lee JK, Kung MC, Kung HH, Mockros LF. Microchannel technologies for artificial lungs: (3) open rectangular channels. ASAIO J 2008;54: 390–395. 57 Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE. Reconstituting organ-level lung functions on a chip. Science 2010;328:1662–1668. 58 Huh D, Leslie DC, Matthews BD, Fraser JP, Jurek S, Hamilton GA, Thorneloe KS, McAlexander MA, Ingber DE. A human disease model of drug toxicity-induced pulmonary edema in a lung-on-achip microdevice. Sci Transl Med 2012;4:ra147. 59 Tschumperlin DJ, Boudreault F, Liu F. Recent advances and new opportunities in lung mechanobiology. J Biomech 2010;43:99–107. 60 Perlman CE, Bhattacharya J. Alveolar expansion imaged by optical sectioning microscopy. J Appl Physiol 2007;103:1037–1044. 61 Liu F, Mih JD, Shea BS, Kho AT, Sharif AS, Tager AM, Tschumperlin DJ. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression. J Cell Biol 2010;190:693–706. 62 Tilghman RW, Cowan CR, Mih JD, Koryakina Y, Gioeli D, Slack-Davis JK, Blackman BR, Tschumperlin DJ, Parsons JT. Matrix rigidity regulates cancer cell growth and cellular phenotype. PLoS ONE 2010;5:e12905. 63 Chen DL, Rosenbluth DB, Mintun MA, Schuster DP. FDG-PET imaging of pulmonary inflammation in healthy volunteers after airway instillation of endotoxin. J Appl Physiol 2006;100: 1602–1609. 64 Osmanagic E, Sukstanskii AL, Quirk JD, Woods JC, Pierce RA, Conradi MS, Weibel ER, Yablonskiy DA. Quantitative assessment of lung microstructure in healthy mice using an MR-based 3He lung morphometry technique. J Appl Physiol 2010;109:1592–1599. 65 Dregely I, Ruset IC, Mata JF, Ketel J, Ketel S, Distelbrink J, Altes TA, Mugler JP III, Wilson Miller G, William Hersman F, et al. Multipleexchange-time xenon polarization transfer contrast (MXTC) MRI: initial results in animals and healthy volunteers. Magn Reson Med 2012;67:943–953. 66 Wang W, Nguyen NM, Yablonskiy DA, Sukstanskii AL, Osmanagic E, Atkinson JJ, Conradi MS, Woods JC. Imaging lung microstructure in mice with hyperpolarized 3He diffusion MRI. Magn Reson Med 2011;65:620–626. 67 Hajari AJ, Yablonskiy DA, Quirk JD, Sukstanskii AL, Pierce RA, Deslee ´ G, Conradi MS, Woods JC. Imaging alveolar-duct geometry during expiration via ³He lung morphometry. J Appl Physiol 2011; 110:1448–1454. 68 Imai F, Kashiwagi R, Imai H, Iguchi S, Kimura A, Fujiwara H. Hyperpolarized 129Xe MR imaging with balanced steady-state free precession in spontaneously breathing mouse lungs. Magn Reson Med Sci 2011;10:33–40. 69 Matsuoka S, Patz S, Albert MS, Sun Y, Rizi RR, Gefter WB, Hatabu H. Hyperpolarized gas MR Imaging of the lung: current status as a research tool. J Thorac Imaging 2009;24:181–188. 70 Mugler JP III, Altes TA, Ruset IC, Dregely IM, Mata JF, Miller GW, Ketel S, Ketel J, Hersman FW, Ruppert K. Simultaneous magnetic

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resonance imaging of ventilation distribution and gas uptake in the human lung using hyperpolarized xenon-129. Proc Natl Acad Sci USA 2010;107:21707–21712. Tanoli TS, Woods JC, Conradi MS, Bae KT, Gierada DS, Hogg JC, Cooper JD, Yablonskiy DA. In vivo lung morphometry with hyperpolarized 3He diffusion MRI in canines with induced emphysema: disease progression and comparison with computed tomography. J Appl Physiol 2007;102:477–484. Woods JC, Choong CK, Yablonskiy DA, Bentley J, Wong J, Pierce JA, Cooper JD, Macklem PT, Conradi MS, Hogg JC. Hyperpolarized 3He diffusion MRI and histology in pulmonary emphysema. Magn Reson Med 2006;56:1293–1300. Yablonskiy DA, Sukstanskii AL, Leawoods JC, Gierada DS, Bretthorst GL, Lefrak SS, Cooper JD, Conradi MS. Quantitative in vivo assessment of lung microstructure at the alveolar level with hyperpolarized 3He diffusion MRI. Proc Natl Acad Sci USA 2002; 99:3111–3116. Schrepfer S, Deuse T, Reichenspurner H, Fischbein MP, Robbins RC, Pelletier MP. Stem cell transplantation: the lung barrier. Transplant Proc 2007;39:573–576. Gao J, Dennis JE, Muzic RF, Lundberg M, Caplan AI. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 2001;169:12–20. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, SemprunPrieto L, Delafontaine P, Prockop DJ. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 2009;5:54–63. Choi H, Lee RH, Bazhanov N, Oh JY, Prockop DJ. Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosaninduced mouse peritonitis by decreasing TLR2/NF-kB signaling in resident macrophages. Blood 2011;118:330–338. Roddy GW, Rosa RH Jr, Oh JY, Ylostalo JH, Bartosh TJ Jr, Choi H, Lee RH, Yasumura D, Ahern K, Nielsen G, et al. Stanniocalcin-1 rescued photoreceptor degeneration in two rat models of inherited retinal degeneration. Mol Ther 2012;20:788–797. Boregowda SV, Phinney DG. Therapeutic applications of mesenchymal stem cells: current outlook. BioDrugs 2012;26: 201–208. Lalu MM, McIntyre L, Pugliese C, Fergusson D, Winston BW, Marshall JC, Granton J, Stewart DJ; Canadian Critical Care Trials Group. Safety of cell therapy with mesenchymal stromal cells (SafeCell): a systematic review and meta-analysis of clinical trials. PLoS ONE 2012;7:e47559. Keating A. Mesenchymal stromal cells: new directions. Cell Stem Cell 2012;10:709–716. Le Blanc K, Mougiakakos D. Multipotent mesenchymal stromal cells and the innate immune system. Nat Rev Immunol 2012;12: 383–396. Bianco P, Cao X, Frenette PS, Mao JJ, Robey PG, Simmons PJ, Wang CY. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med 2013;19:35–42. Prockop DJ, Oh JY. Medical therapies with adult stem/progenitor cells (MSCs): a backward journey from dramatic results in vivo to the cellular and molecular explanations. J Cell Biochem 2012;113: 1460–1469. François M, Galipeau J. New insights on translational development of mesenchymal stromal cells for suppressor therapy. J Cell Physiol 2012;227:3535–3538. Singer NG, Caplan AI. Mesenchymal stem cells: mechanisms of inflammation. Annu Rev Pathol 2011;6:457–478. Prockop DJ, Kota DJ, Bazhanov N, Reger RL. Evolving paradigms for repair of tissues by adult stem/progenitor cells (MSCs). J Cell Mol Med 2010;14:2190–2199. Russell KC, Phinney DG, Lacey MR, Barrilleaux BL, Meyertholen KE, O’Connor KC. In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem Cells 2010;28:788–798.

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89 Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, Phinney DG. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA 2003;100: 8407–8411. 90 Gupta N, Su X, Popov B, Lee JW, Serikov V, Matthay MA. Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. J Immunol 2007;179:1855–1863. 91 Nemeth ´ K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, Doi K, Robey PG, Leelahavanichkul K, Koller BH, Brown JM, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med 2009;15:42–49. 92 Mei SHJ, Haitsma JJ, Dos Santos CC, Deng Y, Lai PFH, Slutsky AS, Liles WC, Stewart DJ. Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis. Am J Respir Crit Care Med 2010;182: 1047–1057. 93 Lee JW, Fang X, Krasnodembskaya A, Howard JP, Matthay MA. Concise review: Mesenchymal stem cells for acute lung injury: role of paracrine soluble factors. Stem Cells 2011;29:913–919. 94 Krasnodembskaya A, Song Y, Fang X, Gupta N, Serikov V, Lee JW, Matthay MA. Antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide LL-37. Stem Cells 2010;28:2229–2238. 95 Lee JW, Fang X, Gupta N, Serikov V, Matthay MA. Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung. Proc Natl Acad Sci USA 2009;106:16357–16362. 96 Loebinger MR, Janes SM. Stem cells as vectors for antitumour therapy. Thorax 2010;65:362–369. 97 Loebinger MR, Kyrtatos PG, Turmaine M, Price AN, Pankhurst Q, Lythgoe MF, Janes SM. Magnetic resonance imaging of mesenchymal stem cells homing to pulmonary metastases using biocompatible magnetic nanoparticles. Cancer Res 2009;69: 8862–8867. 98 Loebinger MR, Eddaoudi A, Davies D, Janes SM. Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer. Cancer Res 2009;69:4134–4142. 99 Jang HJ, Cho KS, Park HY, Roh HJ. Adipose tissue-derived stem cells for cell therapy of allergic airways diseases in mice. Acta Histochemica 2010;113:501–507. 100 Bonfield TL, Nolan Koloze MT, Lennon DP, Caplan AI. Defining human mesenchymal stem cell efficacy in vivo. J Inflamm (Lond) 2010;7:51. 101 Cho KS, Park HK, Park HY, Jung JS, Jeon SG, Kim YK, Roh HJ. IFATS collection: immunomodulatory effects of adipose tissuederived stem cells in an allergic rhinitis mouse model. Stem Cells 2009;27:259–265. 102 Bonfield TL, Koloze MF, Lennon DP, Zuchowski B, Yang SE, Caplan AI. Human mesenchymal stem cells suppress chronic airway inflammation in the murine ovalbumin asthma model. Am J Physiol Lung Cell Mol Physiol 2010;299:L760–L770. 103 Park HK, Cho KS, Park HY, Shin DH, Kim YK, Jung JS, Park SK, Roh HJ. Adipose-derived stromal cells inhibit allergic airway inflammation in mice. Stem Cells Dev 2010;19:1811–1818. 104 Nemeth K, Keane-Myers A, Brown JM, Metcalfe DD, Gorham JD, Bundoc VG, Hodges MG, Jelinek I, Madala S, Karpati S, et al. Bone marrow stromal cells use TGF-beta to suppress allergic responses in a mouse model of ragweed-induced asthma. Proc Natl Acad Sci USA 2010;107:5652–5657. 105 Firinci F, Karaman M, Baran Y, Bagriyanik A, Ayyildiz ZA, Kiray M, Kozanoglu I, Yilmaz O, Uzuner N, Karaman O. Mesenchymal stem cells ameliorate the histopathological changes in a murine model of chronic asthma. Int Immunopharmacol 2011;11: 1120–1126. 106 Goodwin M, Sueblinvong V, Eisenhauer P, Ziats NP, LeClair L, Poynter ME, Steele C, Rincon M, Weiss DJ. Bone marrow-derived mesenchymal stromal cells inhibit Th2-mediated allergic airways inflammation in mice. Stem Cells 2011;29:1137–1148.

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VERMONT STEM CELL CONFERENCE 107 Kapoor S, Patel SA, Kartan S, Axelrod D, Capitle E, Rameshwar P. Tolerance-like mediated suppression by mesenchymal stem cells in patients with dust mite allergy-induced asthma. J Allergy Clin Immunol 2012;129:1094–1101. 108 Kavanagh H, Mahon BP. Allogeneic mesenchymal stem cells prevent allergic airway inflammation by inducing murine regulatory T cells. Allergy 2011;66:523–531. 109 Lee SH, Jang AS, Kwon JH, Park SK, Won JH, Park CS. Mesenchymal stem cell transfer suppresses airway remodeling in a toluene diisocyanate-induced murine asthma model. Allergy Asthma Immunol Res 2011;3:205–211. 110 Ou-Yang HF, Huang Y, Hu XB, Wu CG. Suppression of allergic airway inflammation in a mouse model of asthma by exogenous mesenchymal stem cells. Exp Biol Med (Maywood) 2011;236: 1461–1467. 111 Ionescu LI, Alphonse RS, Arizmendi N, Morgan B, Abel M, Eaton F, Duszyk M, Vliagoftis H, Aprahamian TR, Walsh K, et al. Airway delivery of soluble factors from plastic-adherent bone marrow cells prevents murine asthma. Am J Respir Cell Mol Biol 2012;46: 207–216. 112 Grove DA, Xu J, Joodi R, Torres-Gonzales E, Neujahr D, Mora AL, Rojas M. Attenuation of early airway obstruction by mesenchymal stem cells in a murine model of heterotopic tracheal transplantation. J Heart Lung Transplant 2011;30:341–350. 113 Shigemura N, Okumura M, Mizuno S, Imanishi Y, Nakamura T, Sawa Y. Autologous transplantation of adipose tissue-derived stromal cells ameliorates pulmonary emphysema. Am J Transplant 2006;6: 2592–2600. 114 Shigemura N, Okumura M, Mizuno S, Imanishi Y, Matsuyama A, Shiono H, Nakamura T, Sawa Y. Lung tissue engineering technique with adipose stromal cells improves surgical outcome for pulmonary emphysema. Am J Respir Crit Care Med 2006;174: 1199–1205. 115 Yuhgetsu H, Ohno Y, Funaguchi N, Asai T, Sawada M, Takemura G, Minatoguchi S, Fujiwara H, Fujiwara T. Beneficial effects of autologous bone marrow mononuclear cell transplantation against elastaseinduced emphysema in rabbits. Exp Lung Res 2006;32:413–426. 116 Zhen G, Liu H, Gu N, Zhang H, Xu Y, Zhang Z. Mesenchymal stem cells transplantation protects against rat pulmonary emphysema. Front Biosci 2008;13:3415–3422. 117 Zhen G, Xue Z, Zhao J, Gu N, Tang Z, Xu Y, Zhang Z. Mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-induced emphysematous lungs and inhibits apoptosis of lung cells. Cytotherapy 2010;12:605–614. 118 Hoffman AM, Paxson JA, Mazan MR, Davis AM, Tyagi S, Murthy S, Ingenito EP. Lung-derived mesenchymal stromal cell posttransplantation survival, persistence, paracrine expression, and repair of elastase-injured lung. Stem Cells Dev 2011;20:1779–1792. 119 Katsha AM, Ohkouchi S, Xin H, Kanehira M, Sun R, Nukiwa T, Saijo Y. Paracrine factors of multipotent stromal cells ameliorate lung injury in an elastase-induced emphysema model. Mol Ther 2011;19:196–203. 120 Schweitzer KS, Johnstone BH, Garrison J, Rush NI, Cooper S, Traktuev DO, Feng D, Adamowicz JJ, Van Demark M, Fisher AJ, et al. Adipose stem cell treatment in mice attenuates lung and systemic injury induced by cigarette smoking. Am J Respir Crit Care Med 2011;183:215–225. 121 Ingenito EP, Tsai L, Murthy S, Tyagi S, Mazan M, Hoffman A. Autologous lung-derived mesenchymal stem cell transplantation in experimental emphysema. Cell Transplant 2012;21:175–189.

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122 Kim SY, Lee JH, Kim HJ, Park MK, Huh JW, Ro JY, Oh YM, Lee SD, Lee YS. Mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smoke-induced damage. Am J Physiol Lung Cell Mol Physiol 2012;302:L891–L908. 123 Yoder MC. Progenitor cells in the pulmonary circulation. Proc Am Thorac Soc 2011;8:466–470. 124 Yoder MC. Human endothelial progenitor cells. Cold Spring Harb Perspect Med 2012;2:a006692. 125 Yoder MC. Is endothelium the origin of endothelial progenitor cells? Arterioscler Thromb Vasc Biol 2010;30:1094–1103. 126 Aslam M, Baveja R, Liang OD, Fernandez-Gonzalez A, Lee C, Mitsialis SA, Kourembanas S. Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease. Am J Respir Crit Care Med 2009;180:1122–1130. 127 Chang YS, Oh W, Choi SJ, Sung DK, Kim SY, Choi EY, Kang S, Jin HJ, Yang YS, Park WS. Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats. Cell Transplant 2009;18:869–886. 128 van Haaften T, Byrne R, Bonnet S, Rochefort GY, Akabutu J, Bouchentouf M, Rey-Parra GJ, Galipeau J, Haromy A, Eaton F, et al. Airway delivery of mesenchymal stem cells prevents arrested alveolar growth in neonatal lung injury in rats. Am J Respir Crit Care Med 2009;180:1131–1142. 129 Chang YS, Choi SJ, Sung DK, Kim SY, Oh W, Yang YS, Park WS. Intratracheal transplantation of human umbilical cord bloodderived mesenchymal stem cells dose-dependently attenuates hyperoxia-induced lung injury in neonatal rats. Cell Transplant 2011;20:1843–1854. 130 Pierro M, Ionescu L, Montemurro T, Vadivel A, Weissmann G, Oudit G, Emery D, Bodiga S, Eaton F, Peault ´ B, et al. Short-term, longterm and paracrine effect of human umbilical cord-derived stem cells in lung injury prevention and repair in experimental bronchopulmonary dysplasia. Thorax 2013;68:475–484. 131 Zhang H, Fang J, Su H, Yang M, Lai W, Mai Y, Wu Y. Bone marrow mesenchymal stem cells attenuate lung inflammation of hyperoxic newborn rats. Pediatr Transplant 2012;16:589–598. 132 Zhang X, Wang H, Shi Y, Peng W, Zhang S, Zhang W, Xu J, Mei Y, Feng Z. Role of bone marrow-derived mesenchymal stem cells in the prevention of hyperoxia-induced lung injury in newborn mice. Cell Biol Int 2012;36:589–594. 133 Tropea KA, Leder E, Aslam M, Lau AN, Raiser DM, Lee JH, Balasubramaniam V, Fredenburgh LE, Alex Mitsialis S, Kourembanas S, et al. Bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2012;302:L829–L837. 134 Sutsko RP, Young KC, Ribeiro A, Torres E, Rodriguez M, Hehre D, Devia C, McNiece I, Suguihara C. Long-term reparative effects of mesenchymal stem cell therapy following neonatal hyperoxiainduced lung injury. Pediatr Res 2013;73:46–53. 135 Lavoie JR, Stewart DJ. Genetically modified endothelial progenitor cells in the therapy of cardiovascular disease and pulmonary hypertension. Curr Vasc Pharmacol 2012;10: 289–299. 136 Zhao YD, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ. Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease. Circ Res 2005;96:442–450.

AnnalsATS Volume 10 Number 5 | October 2013